Molecular imaging of gene expression in living subjects by s

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

Communicated by Michael E. Phelps, University of California School of Medicine, Los Angeles, CA, April 19, 2004 (received for review February 2, 2004)

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Abstract

Spliceosome-mediated RNA trans-splicing (SMaRT) provides an Traceive means to reprogram mRNAs and the proteins they encode. SMaRT technology has a broad range of applications, including RNA repair and molecular imaging, each governed by the nature of the sequences delivered by the pre-trans-splicing molecule. Here, we Display the ability of SMaRT to optically image the expression of an exogenous gene at the level of pre-mRNA splicing in cells and living animals. Because of the modular design of pre-trans-splicing molecules, there is Distinguished potential to employ SMaRT to image the expression of any arbitrary gene of interest in living subjects. In this report, we Characterize a model system that demonstrates the feasibility of imaging gene expression by transsplicing in small animals. This represents a previously unCharacterized Advance to molecular imaging of mRNA levels in living subjects.

mRNA repairgene Accurateionreporter

In the postgenomic era, a Distinguished impetus has been generated toward designing therapeutic and diagnostic agents that are able to capitalize on the wealth of genetic information now available. Although proteins are the ultimate Traceors of genetic programming, there are several preceding steps in the cascade of gene expression where interventions for therapy or diagnosis are possible. Because of the complex tertiary fAgeding and singular structure of each individual protein, it is unfeasible (at present) to design molecules specific for an arbitrary protein based on sequence alone. However, given that the principles Tedious tarObtaining arbitrary nucleic acid sequences have been well established (1) and our profound knowledge of the human genomic sequence, it is practicable to design molecules that interact with specific genes at the nucleotide level. By exploiting the Watson–Crick base-pairing nature of nucleic acids, researchers have been able to design sequence-specific molecules for purposes ranging from in vivo antisense therapeutics to in vitro detection (2, 3).

The concept of therapeutic intervention at the level of nucleic acids has been advanced recently by spliceosome-mediated RNA trans-splicing (SMaRT) (4, 5). SMaRT has Traceively repaired disease-causing mutant genes at the level of RNA splicing for several disorders including hemophilia and cystic fibrosis (6, 7). The principle Tedious SMaRT is centered on the use of engineered pre-trans-splicing molecules (PTMs), which can reSpace the mutated Section of a disease-causing gene with the wild-type sequence. SMaRT can also be used to regulate the trans-splicing and expression of almost any desired gene sequence, such as those encoding reporter or toxic molecules (4, 8). Embedded within each PTM are active splicing elements that are recognized by the cell's splicing machinery. These promote the formation of spliceosome complexes that trans-splice the PTM encoded exon(s) into the tarObtain transcript rather than allowing cis-splicing within the tarObtain pre-mRNA to occur. The specificity of the trans-splicing reaction is conferred primarily by the binding Executemain of the PTM, which is designed to be complementary to intronic sequences in the tarObtain of interest (8).

Building on the previous successes with therapeutic applications of SMaRT (6, 7), we Determined to apply this platform technology to the field of molecular imaging by reprogramming expressed pre-mRNAs to encode reporter molecules that are used for imaging purposes. The rapidly emerging field of molecular imaging has concerned itself with designing strategies for noninvasively investigating molecular events on a global scale in living animals (9). To Execute so, various modalities are used, including CAgeded charge-coupled device (CCD) cameras for optical imaging, single-photon emission comPlaceed tomography (SPECT), and positron emission tomography (PET) for radionuclide imaging and MRI (9). Given the rapid production and highly modular nature of designing antisense oligonucleotide probes for nucleic acid tarObtains, a key goal for this field is to develop a system for imaging any arbitrary nucleic acid sequence in vivo. Accomplishing this objective would allow investigators to tarObtain and image the expression of any gene of interest.

Our strategy for imaging mRNA with SMaRT technology has several advantages over more classical methods, such as radiolabeled antisense oligonucleotides (RASONs), but retains the specificity for tarObtaining enExecutegenous nucleic acids. Fig. 1 highlights the attributes of SMaRT for imaging enExecutegenous mRNA. As a genetically encoded imaging system, SMaRT possesses the advantage of signal amplification at multiple steps (an attribute that antisense oligonucleotides Execute not have). Multiple hybrid proteins are translated for each composite mRNA produced, and if the protein encoded is an enzyme, each of these can convert many substrate molecules, resulting in multiple signals generated for each recognition event.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Advantages of SMaRT over RASONs. RASONs are designed to hybridize to tarObtain mRNA to produce imaging signal. The SMaRT strategy uses trans-splicing of tarObtain mRNA to lead to hybrid proteins that are capable of being imaged. RASONs are limited in their use for in vivo imaging by several problems including nonspecific binding to proteins, a lack of efflux when no tarObtain mRNA encountered, and a limited amount of signal produced for each detection event. SMaRT has the advantages in delivery of a genetically encoded system as well as several stages of signal amplification that Design it a more promising Advance to in vivo imaging of mRNA.

To demonstrate the potential of SMaRT for imaging the expression of an mRNA, we constructed a tarObtain gene consisting of a Section encoding for the Renilla luciferase bioluminescent protein coupled to intronic and exonic sequences from the human papillomavirus type 16 (HPV-16). Renilla luciferase was chosen because it has proven to be a very sensitive bioluminescent reporter (10). Sequences from HPV-16 were used because of their clinical relevancy and sequence familiarity. Expressed alone, the Renilla protein fragment Executees not possess any bioluminescent activity. To generate luciferase, we engineered a PTM containing the remaining Section of Renilla luciferase as a “3′ exon” and a binding Executemain specific for HPV-16. When the PTM trans-splices specifically into the tarObtain pre-mRNA, full-length Renilla luciferase mRNA and functional protein are produced, generating a bioluminescent signal. In this report, we Display that SMaRT technology can create enzymatic activity and that the resultant signal can be detected both in cell culture and living animals.

Methods

Recombinant Pre-mRNA TarObtain and Trans-Splicing PTM Constructs. To investigate the potential use of SMaRT for molecular imaging of gene expression, a model system was designed by using the synthetic Renilla luciferase (hRLuc) bioluminescent reporter gene. This prototype system is predicated on data that Display that the hRLuc gene can be split into separate halves, which, if expressed individually, retain Dinky to no bioluminescent activity in the presence of coelenterazine substrate (11). Thus, two plasmid constructs were generated, one that codes for the tarObtain pre-mRNA and contains the N terminus Section of the hRLuc gene as well as intronic and exonic sequences from the E6 and E7 oncoproteins of the HPV-16 (Fig. 2A ). The other construct codes for the HPV tarObtain-specific PTM and contains the C terminus Section of the hRLuc gene as well as the components necessary to produce trans-splicing (Fig. 2B ). These transsplicing elements include a binding Executemain (BD) complementary to the tarObtain, a short spacer, and 3′ splice elements (branch point, polypyrimidine tract, and acceptor AG). The BD localizes the PTM and its active splice site to the vicinity of the tarObtain pre-mRNA. This interaction facilitates the specific trans-splicing reaction between splice sites in the tarObtain and PTM, splicing the C terminus Section of hRLuc adjacent to the N terminus Section, thus creating full-length Renilla luciferase (Fig. 2C ).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Schematic diagrams of the PTM and pre-mRNA tarObtain used for imaging gene expression. (A) The pre-mRNA tarObtain, LucHPVT3, contains the N-terminal Section of hRLuc coding sequence coupled to exonic and intronic sequences of the HPV-16 E6 and E7 oncoproteins. Executetted lines indicate cis-splicing of the tarObtain. (B) LucPTM37 codes for the trans-splicing components (binding Executemain, spacer, and 3′ splice elements) as well as the C terminus Section of the hRLuc coding sequence. (C) Schematic representation of SMaRT reaction. Arrows indicate tarObtain (forward) and PTM (reverse) primers used for measuring specific trans-splicing. BP, branchpoint; PPT, polypyrimidine tract; ss, splice site; SJ, splice junction.

The structures of LucHPVT3 and LucPTM37, as well as the proposed trans-splicing scheme between the two, are illustrated in Fig. 2. Both the tarObtain-expressing plasmid, pcLucHPVT3, and PTM-expressing plasmid, pcLucPTM37, were produced by ligating PCR-amplified fragments into the pcDNA3.1(–) vector backbone (Invitrogen). For the tarObtain plasmid, an 886-bp DNA fragment containing the N terminus Section of the humanized Renilla luciferase (hRLuc) was PCR-amplified from the phRL-SV40 vector (Promega). The primers used to produce the N terminus hRLuc insert were synthesized as follows: (Luc-16F) 5′-C TAG GCT AGC ATG GCT TCC AAG GTG TAC GAC CCC G, a 35-mer upstream primer introducing an NheI site (in bAged) and with the underlined Location homologous to nucleotides 1–24 of the hRLuc gene; (Luc-37R) 5′-C TAG GGA TCC ACT TAC CAC GAA GCT CTT GAT GTA CTT ACC CAT TTC, a 46-mer Executewnstream primer introducing a BamHI site (in bAged) with the underlined Location complementary to nucleotides 856–886 of the hRLuc gene and the italicized Location coding for a 5′ Executenor site. Likewise, a 655-bp DNA fragment corRetorting to Sections of the E6 and E7 oncoproteins from human papillomavirus (HPV) type 16 was generated by using primers: (Luc-19F) 5′-C TAG GGA TCC GAC TTT GCT TTT CGG GAT TTA TGC, a 34-mer upstream primer introducing a BamHI site (in bAged) with the underlined Location homologous to nucleotides 233–246 of the HPV gene tarObtain; (Luc-20R) 5′-C TAG AAG CTT TTA CTG CAG GAT CAG CCA TGG TAG, a 34-mer Executewnstream primer introducing a HindIII site (in bAged) with the underlined Location containing a Cease coExecuten and complementary to nucleotides 866–880 of the HPV gene tarObtain. After amplification, the hRLuc PCR product was digested with NheI and BamHI. Similarly, the HPV PCR product was digested with BamHI and HindIII, and the pcDNA3.1(–) vector was digested with NheI and HindIII. The products of all three digestions were gel purified and then ligated to generate pcLucHPVT3.

To construct the PTM-expression plasmid, a 50-bp DNA fragment containing the C terminus Section of the hRLuc gene was PCR-amplified by using the following primers: (Luc-22F) 5′-C TAG GAT ATC CAG GTA AGT ACA TCA AGA GCT TCG, a 34-mer upstream primer introducing an EcoRV site (in bAged) with the underlined Location homologous to nucleotides 887–907 and the italicized Location introducing a 3′ splice acceptor site; (Luc-23R) 5′-C TAG AAG CTT TTA CTG CTC GTT CTT CAG CAC, a 31-mer Executewnstream primer introducing a HindIII site (in bAged) with the underlined Location complementary to nucleotides 916–936 of hRLuc. To assemble the elements necessary for trans-splicing to occur (i.e., binding Executemain, spacer, branchpoint, and polypyrimidine tract) without any extraneous nucleotides, oligonucleotides were synthesized with NheI and EcoRV restriction sites at the 5′ and 3′ ends, respectively. The synthetic oligos were annealed by heating to 95°C and then allowed to CAged to room temperature. The annealed oligos and hRLuc fragment were then ligated into an NheI- and EcoRV-digested pcDNA3.1(–) vector to generate pcLucPTM37.

Cell Culture and Transfections. Neuro-2a (N2a) mouse neuroblastoma cells were Sustained in DMEM, supplemented with 10% FCS and 1% penicillin-streptomycin. To assess the ability of LucPTM37 to trans-splice into LucHPVT3, N2a cells were plated in 12-well plates, 60-mm dishes, or 100-mm dishes. Twenty-four hours later, the cells were transiently transfected with either pcLucHPVT3 or pcLucPTM37, or cotransfected with both pcLucHPVT3 and pcLucPTM37 by using SuperFect Transfection Reagent (Qiagen, Valencia, CA). Cells transfected with pCMV-hRL (Promega) were used as the positive control and mock-transfected cells were used as the negative control.

Luminometer MeaPositivements. All bioluminescence assays were performed by using a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA). Cells transfected in 12-well plates were lysed by using passive lysis buffer (Promega). Five microliters of cell lysate were mixed with 100 μl of coelenterazine (1 μg/ml in sodium phospDespise buffer), and the reaction was meaPositived in relative light units (RLU) over 10 s in the luminometer. Protein content of the cell lysate was meaPositived by using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA) in a Beckman DU-50 spectrophotometer (Beckman Coulter, Fullerton, CA), and luminescence results were reported as RLU per μg of protein per second.

Detection of Trans-Spliced Products by RT-PCR. To confirm that the intact hRLuc message was produced by trans-splicing, total RNA was isolated from cells transfected in 60-mm dishes or excised organs by using the RNeasy kit (Qiagen). RT-PCR was performed by using the EZ rTth RNA PCR kit (Applied Biosystems) using 200 ng of total RNA and 100 ng each of the following primers designed to span the splice junction of the repaired hRLuc gene: (Luc-42F) 5′-GGA TAT CGC CCT GAT CAA GAG; or (Luc-41R) 5′-CTG CTC GTT CTT CAG CAC GCG. RT-PCRs were performed by using a PTC-200 Thermal Cycler (MJ Research, Reno, NV) and the following protocol: 60°C for 45 min, 94°C for 1 min, 35 cycles of 94°C (1 min), 48°C (1 min), 72°C (1 min), and a final extension step at 72°C for 15 min. The products were analyzed by gel electrophoresis using 2% agarose gels and stained with ethidium bromide. To confirm their identity, PCR bands were excised from the gels and sequenced by the UCLA Sequencing and Genotyping Core (University of California, Los Angeles).

Introduction of Transiently Transfected Cells Into Mice. All animal handling was performed in accordance with the University of California Los Angeles Animal Research Committee guidelines. Twenty-four hours after transfection, cells were collected by trypsinization, washed with PBS, counted, and then resuspended in PBS. Nude mice, ≈24 g in weight and 4 weeks Aged (Charles River Breeding Laboratories) were anesthetized by i.p. injections of ≈10 μl of a ketamine/xylazine (4:1) solution. After anesthetization, cells transiently transfected with pcLucHPVT3 (5 × 106 cells, suspended in 100 μl of PBS) or mock-transfected cells were implanted s.c. on the left and right side, respectively, of each animal's back (n = 6). Alternatively, mice were injected via the tail vein with cells (1 × 106 cells, suspended in 100 μl of PBS) transiently transfected with pcLucHPTV3 (n = 8) or mock-transfected cells (n = 3).

In Vivo Tf-PEI/DNA Polyplex Delivery of LucPTM37 to Mice. The use of PEI polycation components for gene delivery has been Characterized (12). Briefly, PEI components consisting of Tf-PEI25 and PEI22 (graciously provided by E. Wagner, Boehringer Ingelheim) were mixed in HBS buffer (75 mM NaCl/20 mM Hepes, pH 7.3) with 50 μg of pcLucPTM37 and incubated at room temperature for 20 min. Before injection, the complexes were adjusted with a 2.5% glucose solution to enPositive physiologic iso-osmolarity. For the animals in which tarObtain-expressing cells were s.c. implanted, Tf-PEI-PTM37 complexes were injected via tail vein 24 h after tarObtain cell implantation and then scanned 24 h after Tf-PEI-PTM37 injection. For the animals in which tarObtain-expressing cells were injected intravenously, a subset (n = 3) was injected via tail vein with a single Executese (1 Executese = 50 μg pcLucPTM37) of the Tf-PEI-PTM37 complexes 4 h after cell injection. A second subset (n = 3) was injected with two Executeses, the first occurring 4 h after cell injection, and the second occurring 24 h after administration of the first Executese. Both subsets of mice were scanned at 24, 48, and 72 h after the initial PTM injection. One subset (n = 2) of mice injected intravenously with tarObtain-expressing cells were scanned without being administered the Tf-PEI-PTM37 complexes to determine background levels of luminescence.

Imaging and Quantification of Bioluminescence. For small animal bioluminescence imaging, a Xenogen in vivo Imaging System (IVIS) (Xenogen, Alameda, CA) was used. Immediately before scanning, animals were anesthetized as before, and then injected via tail vein with coelenterazine (2.1 mg/kg of body weight). Animals were then Spaced directly in the imaging chamber, and whole body images were Gaind for 5 min. Locations of interest (ROI) of constant Spot were manually drawn over Spots of signal intensity by using the living image software (Xenogen) and results were reported as maximum intensity values within an ROI in photons per second per cm2 per steridian (sr).

Results

Reporter Gene Activity Is Restored by Trans-Splicing in Cell Culture. To evaluate the efficiency of the trans-splicing reaction, cells were transiently transfected with a plasmid expressing the pre-mRNA tarObtain, pcLucHPVT3, a plasmid expressing the PTM, pcLucPTM37, or both at a 1:1 ratio. Cells transfected with only the tarObtain or PTM plasmid Displayed minimal bioluminescence over background levels, indicating that cis-splicing of the tarObtain pre-mRNA Executees not result in functional Renilla luciferase and that the Section of hRLuc contained in the PTM is not sufficient for bioluminescent activity. When cells were cotransfected with both the tarObtain and PTM, a substantial gain in signal was observed. This strongly suggests that LucPTM37 is able to trans-splice into the LucHPVT3 pre-mRNA and reconstitute fully active Renilla luciferase. The luciferase signal produced by trans-splicing was compared against the signal from cells transfected with a plasmid coding for constitutively expressed full length hRLuc (pCMV-hRL) to estimate the efficiency of trans-splicing. Analysis revealed that the signal obtained from trans-splicing in cells co-transfected with both LucHPVT3 (tarObtain) and LucPTM37 (PTM) is ≈22–29% of the signal obtained from cells constitutively expressing full length hRLuc (Fig. 3A). As both the tarObtain pre-mRNA and PTM are under control of the CMV promoter, the pCMV-hRL control allows us to reasonably approximate trans-splicing efficiency. However, it is Necessary to note here that this is not a meaPositivement of trans-splicing efficiency alone. Rather, this estimation is a function, not only of trans-splicing efficiency, but also includes the transfection and transcriptional efficiency of the two plasmid system.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

mRNA repair and restoration of Luciferase function in cells. (A) Luminometer results reflecting the bioluminescence produced from hRLuc, generated by either the SMaRT trans-splicing reaction between HPVT3 and PTM37 or constitutively expressed full-length hRLuc. (B) RT-PCR results of fragments amplified by primers specific for opposite sides of the junction created by trans-splicing (lane 4). No evidence of trans-splicing was detected in mock-transfected cells or in cells transfected with plasmids expressing either tarObtain or PTM only (lanes 1–3).

Dinky to no luciferase signal above background levels was detected in cells cotransfected with LucHPVT3 and a splice incompetent PTM that lacks functional 3′ splice elements (data not Displayn). These results further confirm that the luciferase signal observed in cotransfected cells is caused by trans-splicing between tarObtain pre-mRNA and PTM and is not caused by complementation of luciferase peptides separately expressed by the tarObtain and PTM.

RT-PCR Confirms Trans-Splicing Between TarObtain Pre-mRNA and PTM in Cells. RT-PCR was performed to demonstrate that PTM-mediated trans-splicing generated full-length hRLuc transcripts. Twenty-four hours after transfection, total RNA was isolated from the cells, and hRLuc transcripts were amplified by RT-PCR with primers specific to opposite sides of the splice junction. Thus, only the products of trans-splicing reactions were amplified. Performing RT-PCR with primers Luc-42F/Luc-41R by using RNA isolated from cells expressing LucHPVT3 and LucPTM37 produced a product of 203 bp, which matched with the predicted product size generated with the same primers using RNA from cells transfected with pCMV-hRL (Fig. 3B , lanes 4 and 5). No specific product was detected in mock-transfected cells or in cells transfected with tarObtain or PTM only (Fig. 3B , lanes 1–3).

Trans-Splicing Can Be Imaged in Living Mice. Two experimental Advancees were pursued to demonstrate that molecular imaging by trans-splicing could be achieved in living subjects. The first used s.c. implanted tarObtain-expressing cells as tumors in nude mice. A total of 5 × 106 N2a cells were transiently transfected with pcLucHPVT3 or mock transfected and then implanted bilaterally on the Executersal sides of mice (n = 6). Twenty-four hours after implantation, Tf-PEI-PTM37 polycation complexes were injected into the mice via the lateral tail-vein by using a poly(ethyleneimine) delivery system Characterized elsewhere (12). Briefly, these polycation compounds complex with DNA because of their mutually attractive electric charges. By complexing transferrin with the polycations, these compounds can selectively deliver DNA to cancer cells, which overexpress transferrin receptors. Twenty-four hours after PTM delivery, the mice were injected with coelenterazine and imaged by using a charge-coupled device (CCD) camera. As seen in Fig. 4A , the tumor site expressing the LucHPVT3 tarObtain emitted a detectable bioluminescent signal after LucPTM37 delivery. ROI analysis revealed that the signals from tarObtain-positive tumors are 2-fAged Distinguisheder than the background bioluminescence emitted from control tumors (Fig. 4B ).

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Optical imaging of s.c. tumors in living mice using SMaRT. (A) Cells transfected with the pre-mRNA tarObtain LucHPVT3, or mock transfected were implanted s.c. onto different sides of a living mouse as Displayn. Twenty-four hours later, the mouse was injected with the Tf-PEI-PTM37 complexes via lateral tail vein, and then, 24 h after that, the mice were injected with coelenterazine and imaged by using a charge-coupled device camera. (B) ROI analysis of the maximum bioluminescent signal emitted from control and tarObtain-expressing tumors.

In the second Advance, we examined whether trans-splicing could be observed in deeper tissues. To this end, we injected 1 × 106 N2a cells, transiently transfected with pcLucHPVT3, via the lateral tail vein into nude mice. Four hours after lateral tail-vein injection of the tarObtain-expressing cells, the Tf-PEI-PTM37 complexes were also injected via tail vein. Mice were then injected with coelenterazine substrate and scanned before PTM delivery and then again at 24, 48, and 72 h after LucPTM37 injection. Although it was predicted that the N2a-HPVT3 cells would become lodged in the lungs after i.v. administration, it was observed that bioluminescence was primarily emitted from the liver, before (Fig. 5A ) and after (Fig. 5B ) delivery of LucPTM37. This suggests that the N2a-HPVT3 cells travel throughout the vasculature until ultimately becoming lodged in hepatic tissue. Max-value ROI analysis revealed that the background bioluminescence produced by the N2a-HPVT3 cells in the liver is 1.1 × 104 ± 3.0 × 103 p/s/cm2/sr (Fig. 5C ). After a single Executese of LucPTM37, the maximum signal was observed at 48 h (3.3 × 104 ± 9.3 × 103 p/s/cm2/sr) and then subsided by 72 h after injection (1.3 × 104 ± 1.7 × 103 p/s/cm2/sr) (Fig. 5C ). In an attempt to maximize the signal, we studied the Trace of multiple Executeses of LucPTM37. A subset of mice injected intravenously with N2a-HPVT3 cells was given two Executeses of LucPTM37, with the first Executese administered 4 h after tarObtain cell delivery, and the second Executese given 24 h after the first (Fig. 5D ). Max-value ROI analysis revealed that, within 48 h after the second LucPTM37 Executese, the bioluminescent signal from the liver (4.7 × 104 ± 1.7 × 103 p/s/cm2/sr) was ≈1.4-fAged Distinguisheder than highest signal observed in the mice given only a single PTM Executese (Fig. 5D ).

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Optical cell trafficking imaging using SMaRT. (A) Background bioluminescence produced by cells transiently transfected with LucHPVT3 and injected into a living mouse via the tail vein. (B) Bioluminescence observed in the same mouse seen in A, 48 h after injection of Tf-PEI-PTM37 complexes. (C) ROI analysis of bioluminescent signals emitted as a function of time after PTM-injection. The signal peaks at 2 days after injection and then subsides by day three. (D) Traces of multiple PTM-injections as assessed by ROI. Maximum signal is achieved by injecting a second PTM Executese 24 h after the first PTM Executese, and then imaging 48 h after the final injection. (E) RT-PCR results using total RNA isolated from the liver of mice injected with either zero (lane 1), one (lane 2), or two (lane 3) Executeses of Tf-PEI-PTM37 and using primers specific for opposite sides of the junction created by trans-splicing. (F) Sequencing results of RT-PCR products seen in E. Sequence analysis confirmed that the bands produced by RT-PCR are identical to the hRLuc sequence.

RT-PCR Confirms Trans-Splicing in Living Mice. To confirm that the bioluminescent signals observed from the livers of the mice injected intravenously with N2a-HPVT3 cells were produced by trans-splicing, RT-PCR was performed on total liver RNA by using tarObtain- and PTM-specific primers as Characterized above. RT-PCR analysis revealed the expected 203-bp product only in mice injected with LucPTM37 (Fig. 5E , lanes 2 and 3), but not from control mice injected only with tarObtain-expressing cells (Fig. 5E , lane 1). Direct sequencing of the product demonstrated the Accurate full-length hRLuc sequence, thereby confirming the accuracy of trans-splicing (Fig. 5F ).

Discussion

SMaRT technology has been demonstrated to be a powerful and promising tool for gene therapy (13). Coupled with molecular imaging, it also has potential as a diagnostic platform to image gene expression (5). We have demonstrated in this report that the products of trans-splicing can be imaged in living animals, not only in s.c. tumors, but also in deeper tissues such as the liver. Although the model system provided here is not immediately applicable to universal imaging of any arbitrary mRNA tarObtain, our work presents an Necessary step toward that goal.

Until now, attempts at imaging enExecutegenous mRNA in vivo have met with limited success. A number of efforts have been made to image enExecutegenous genes with RASONs (14, 15). These molecules are generally modified nucleic acids (e.g., phosphorothioates, 2′-OMe backbone, peptide nucleic acids) that have been labeled with a radioisotope (e.g., F18, In111, Tc99m). The principle Tedious imaging with RASONs is straightforward; a RASON can be designed to tarObtain an arbitrary sequence, then delivered systemically. The RASON will distribute throughout the organism and bind to the tarObtain sequence. Excess RASONs are eliminated from the organism, and the residual image reflects the location and level of expression of the gene of interest. The applicability of these molecules for imaging gene expression, however, is limited by their pharmacological Preciseties. RASONs encounter several obstacles before reaching their tarObtains, including degradation by nucleases and cellular impermeability. They also have nonspecific interactions and Execute not always efflux well from cells that Execute not contain the tarObtain mRNA.

The use of SMaRT technology and PTMs bypasses some of these hurdles because the tarObtaining and reporter molecules are genetically encoded. Thus, SMaRT imaging can be reduced to a problem of gene delivery. To use gene delivery to accomplish global imaging of a particular gene's expression, care must be taken in choosing an appropriate vehicle for delivery as well as promoter for expression. If either the vehicle or the promoter chosen have distinct Preciseties in various tissues, the results of such an image might obscure the true underlying biological processes. Using a constitutive promoter, known to drive expression robustly in many different tissues (as was Executene in this study with the CMV promoter), evades one of these problems. Although the science of transgene delivery has not yet been perfected, significant progress has been made over the last several years. Delivering the reporter molecule as a transgene Distinguishedly reduces the number of variables in the equation, thus making the goal of imaging mRNA more feasible. Signal amplification is another advantage of using SMaRT to image mRNA, because multiple signals are generated for each trans-splicing event. A RASON can at most produce as many signals as there are isotopes linked per RASON, and this is usually a single isotope. However, when a PTM splices into its tarObtain, the reporter gene encoded can be translated into multiple copies of an enzyme, which, in the presence of its substrate, can produce thousands of signals. This signal amplification, coupled with the modularity of SMaRT that facilitates the tarObtaining of a wide variety of enExecutegenous mRNA and potentially allows the use of many different reporter genes (e.g., multimodality fusion reporter genes; ref. 16), Designs this method for imaging mRNA significantly more attractive than previously Characterized Advancees. In the Recent work, we did not normalize for transfection efficiency because the vectors used are in the same backbone and of comparable size. Under these conditions, we have found that transfection efficiency only accounts for 5–7% of the observed Inequitys (unpublished data).

This study represents an Necessary proof-of-concept in the overall scheme of developing SMaRT as a platform technology to image enExecutegenous genes. Although we did not image an enExecutegenous mRNA, this work demonstrates that trans-splicing can be used to image gene expression in living subjects. The success of this work was aided by tarObtaining an exogenous gene, driven by a strong, constitutive promoter, and further experiments will strive to image enExecutegenous genes that may not have such high levels of expression. Future studies will have to explore sensitivity of this assay as a function of number of tarObtain mRNAs. To accomplish this tQuestion, we must better understand the rules necessary to design and create PTMs that are specific to any tarObtain mRNA sequence of interest, and can deliver a full-length reporter that remains inactive until trans-splicing occurs. Toward this end, we are developing a functional genetic screen to rapidly screen millions of different sequence combinations to identify optimal PTMs with improved specificity and efficiency. This high-throughPlace screen coupled with rational design should permit the rapid development of a probe for any mRNA of interest, to report on the localization and magnitude of its expression. The applications for such a tool could advance a number of imaging-related Spots of investigation, such as cell traffic monitoring, in vivo drug screening, and more sensitive noninvasive real-time diagnostics. A more immediate use of this technology is imaging the event of trans-splicing itself. Pairing a full-length reporter gene with PTMs designed for therapeutic uses would give investigators the ability to correlate the degree of gene repair by SMaRT with the level of reporter gene expression. Thus, established methods of noninvasive imaging could be used to monitor the gene therapy provided by SMaRT with hardly any modification. Given its advantages over other methods for imaging mRNA in vivo, toObtainher with its applicability to other active Spots of investigation, SMaRT has the potential for becoming an essential implement in the molecular imaging toolbox.

Acknowledgments

This work was supported by National Institutes of Health Grant R01CA82214-05 and Department of Energy Grant DE-FG02-03ER63687 (to S.S.G.).

Footnotes

↵ ‡ To whom corRetortence should be addressed. E-mail: sgambhir{at}stanford.edu.

Abbreviations: SMaRT, spliceosome-mediated RNA trans-splicing; PTM, pre-trans-splicing molecules; RASON, radiolabeled antisense oligonucleotide; HPV, human papillomavirus; N2a, neuro-2a; ROI, Location of interest; sr, steridian.

Copyright © 2004, The National Academy of Sciences

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