A Workflow for In Vivo Evaluation of Candidate Inputs and Outputs for

Publication Date (Web): December 19, 2017. Copyright © 2017 American Chemical Society. *E-mail: [email protected]., *E-mail: ...
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A Workflow for In Vivo Evaluation of Candidate Inputs and Outputs for Cell Classifier Gene Circuits Margaux Dastor, Joerg Schreiber, Laura Prochazka,† Bartolomeo Angelici, Jonathan Kleinert, Ina Klebba,‡ Jiten Doshi, Linling Shen,* and Yaakov Benenson* Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland S Supporting Information *

ABSTRACT: Cell classifier gene circuits that integrate multiple molecular inputs to restrict the expression of therapeutic outputs to cancer cells have the potential to result in efficacious and safe cancer therapies. Preclinical translation of the hitherto developments requires creating the conditions where the animal model, the delivery platform, in vivo expression levels of the inputs, and the efficacy of the output, all come together to enable detailed evaluation of the fully assembled circuits. Here we show an integrated workflow that addresses these issues and builds the framework for preclinical classifier studies using the design framework of microRNA (miRNA, miR)-based classifier gene circuits. Specifically, we employ HCT-116 colorectal cancer cell xenograft in an experimental mouse metastatic liver tumor model together with Adeno-associated virus (AAV) vector delivery platform. Novel engineered AAV-based constructs are used to validate in vivo the candidate inputs miR-122 and miR-7 and, separately, the cytotoxic output HSV-TK/ganciclovir. We show that while the data are largely consistent with expectations, crucial insights are gained that could not have been obtained in vitro. The results highlight the importance of detailed stepwise interrogation of the experimental parameters as a necessary step toward clinical translation of synthetic gene circuits. KEYWORDS: cell classifier circuits, colorectal cancer, mouse tumor model, miRNA, selective cell targeting

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ell classifier gene circuits1 gained traction in recent years as a promising tool to target diseased cells selectively without harmful side effects on standby healthy cells, a “holy grail” in treating many diseases including cancer. This is achieved by combinatorial integration of multiple disease- and tissue-associated molecular markers to elevate the activity of a therapeutic output in diseased cells and inactivate it in healthy cells.2 We have recently shown that this concept is supported, at least in theory, by available gene expression data, and that sets of markers integrated logically in a gene circuit, rather than single selective markers, are required for this task.3 Among the inputs processed by published classifier circuits are endogenous miRNAs,4 transcription factors (TFs),5 and promoter activities.6 MiRNA are conserved small non coding RNAs that regulate gene expression7−9 via base pair binding10 to fully or partially complementary sequences in the target mRNA.11−13 MiRNA are expressed differentially in different tissues10,14,15 and cell types;16,17 they control both normal physiological18−20 and pathological processes,21−23 including cancer. Accordingly, tissue-specific miRNA activity has been exploited to improve the specificity of systemically delivered gene therapy and target transgene expression to certain cell types despite broad tropism of the viral vectors,24−27 as well as restrict the lytic cycle of oncolytic viruses to cancer cells.28 © XXXX American Chemical Society

In recent years we developed multi-input miRNA-based logic circuits to selectively identify and destroy cancer cells4,29,30 based on a combination of both tumor- and tissue-specific miRNA inputs. The circuits comprise a panel of genetically encoded sensors that elicit regulatory effect on the output gene: tumor-specific miRNA inputs indirectly induce the gene by targeting this gene’s repressor, whereas tissue-specific miRNA inputs target the gene directly via complementary sites in the 3′-untranslated region (UTR). While sophisticated circuitry has shown efficacy in vivo in the context of CAR-T cell therapy31−33 and metabolic disease,34 cell classifiers that require cytoplasmic inputs have only been tested in cell culture.6,35 Preclinical translation of mammalian cell classifiers is challenging because many factors need to come together: the presumptive clinical scenario and the respective animal tumor model should be compatible with the chosen method of in vivo delivery; further, the expression of the candidate inputs must be confirmed in vivo, preferably at a single-cell level; and last, the intrinsic antitumor efficacy of the candidate therapeutic outputs should likewise be confirmed in the specific experimental setting. Rational preclinical translation of cell classifiers is difficult unless all these parameters are determined. Received: August 22, 2017

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Figure 1. Setting up the in vivo model. (a) Schematic workflows of the intrasplenic HCT-116LC cell inoculation in NSG mice, in vivo tumor followup by bioluminescent imaging, and tumor and liver tissue characterization at termination. (b) Schematic representation of the HCT-116LC stable cell line generation using TALEN genome editing.51−53 The schemes on top show the DNA constructs used for the cell line generation, with individual functional elements indicated. AAVS1, homology arm for genome integration of the construct in the AAVS1 locus in chromosome 19 (position 19q13.42); NLS, nuclear localization signal. T2A, 2A peptide from Thosea asigna virus capsid protein allowing biscitronic expression of Luciferase and mCitrine. The two constructs containing TALEN R/L53 refer to the transcription activator-like effector nuclease technology B

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components for genome integration. The flow cytometry and imaging data correspond to the HCT-116LC clone used for tumor model establishment. Fluorescent channels, exposure time and objective magnification are shown. (c) The relationship between the luciferase activity, yellow fluorescent protein (mCitrine) expression, and cell numbers for the HCT-116LC cells. Each bar represents the mean bioluminescence signal (ph/s/sr) from a triplicate measurement and the error bars represent one standard deviation. Inset graph shows a linear correlation between the bioluminescence signal and the cell numbers. For each cell number, the corresponding fluorescence micrographs of the yellow fluorescent protein (mCitrine) expression by HCT-116LC is placed underneath the bar chart, scale bars represent 100 μm. mCitrine expression is shown for illustration and it was not quantified. (d) Tumor growth curves showing the in vivo intensity and kinetics of luciferase activity measured by whole body bioluminescence imaging after inoculation of 3 × 105 HCT-116LC cells in NSG mice. Each curve represents data obtained from one animal, as indicated in the legend. The animals were terminated at different time points to examine tumor morphology. (e) Fluorescence micrographs from fresh HCT-116LC (mCitrine-positive nodules) tumor-bearing liver tissue obtained post-termination at indicated time. IDs of the host animals and the waiting time between inoculation and termination are shown (the color code corresponds to panel d). Two sets of image processing parameters, indicated as high and low LUTs (Look Up Table), are used to show the tumor load of the liver tissues. The high LUT is used to visualize large tumors at later time points while the low LUT is used to visualize small tumors at early time points by enhancing the weak signal observed at this stage of tumor development. Scale bars represent 100 μm. (f) Whole body bioluminescence images obtained from a representative animal over time. The color code indicates photon flux (Ph/s/sr).

Among the various challenges listed above, single cell in vivo profiling of candidate classifier inputs stands out as the key prerequisite for the implementation of selective circuits. While the tumor can in principle be profiled in vitro, there is no guarantee that the expression would remain unchanged in vivo. In the case of miRNA classifiers, the experiment should confirm that the tumor-specific inputs are indeed sufficiently active in the tumors in vivo at a single-cell level, while the tissue-specific inputs are active in the cells of the respective tissues without being present or active in the tumors. Current tools for in vitro miRNA profiling comprise bulk characterization using either microarray36 or RNA deep sequencing15,37,38 that result in a loss of singe-cell resolution, although recently a single-cell miRNA sequencing method has been described.39 In vitro single-cell characterization of miRNA activity usually relies on fluorescent reporters that are coexpressed with an internal control gene to normalize for experimental variation; such constructs are encoded in plasmids40−43 as well as in viral vectors.44 The in vivo reporters described so far include an adenoviral vector encoding a luciferase with a miRNA target26 and a fluorescent reporter delivered using a lentiviral vector.45 The luciferase reporters result in a loss of single-cell resolution; the lentiviral fluorescent reporter, while providing single-cell data, is an integrating vector that may not reflect the activity levels obtained with nonintegrating vectors that are the likely candidates for in vivo delivery. Further, to the best of our knowledge, these and other constructs shown so far in vivo do not possess an internal expression control reporter, thus making the results difficult to interpret due to their sensitivity to variations in the experimental conditions such as the amount of the injected reporter vector and other factors that might influence the absolute amount of the delivered reporter rather than the relative knockdown. In this report we describe a case study that builds the required experimental framework for the preclinical translation of miRNA-based cell classifiers in the context of an animal model of a disseminated colorectal cancer metastasis in the liver and an Adeno-associated virus (AAV) vector delivery platform by in vivo validation of the prospective inputs on one hand, and in vivo confirmation of the intrinsic output efficacy, on the other. The candidate inputs include miRNA-122 and miR-7 as respective liver- and tumor-specific markers. The candidate output is an HSV-TK gene in combination with a prodrug ganciclovir (GCV).46 We find that miRNA activity can be reliably and quantitatively assessed simultaneously in the tumor and in healthy tissues, and we also find evidence of mutual

miRNA enrichment between the tumor and the surrounding healthy tissue. In addition we confirm the antitumor efficacy of the HSV-TK/GCV combination in this particular experimental setting. The constructs reported here and the accompanying workflow can be used in similar endeavors to provide a starting point for the design and preclinical evaluation of tumortargeting cell classifiers.

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RESULTS Setting up the In Vivo Model. For the purpose of this study, we have adapted the experimental liver metastasis model of human colon cancer47 established via intrasplenic injection of HCT-116 human colorectal cancer cells in immunecompromised NOD SCID Gamma (NSG) mice; a similar model has already been used for microbial-based cancer therapy delevopment.48,49 The identification of tumors colonizing the healthy tissues is facilitated by the incorporation of a stable fluorescent marker in the tumor cells, enabling their analysis by imaging and flow cytometry (Figure 1a). In parallel, real time tracking of tumor progression requires stable expression of luciferase.50 To achieve these goals we generated a stable HCT116 cell line (HCT-116LC) using a bicistronic construct expressing both luciferase and mCitrine reporters (Figure 1b) integrated with the help of TALEN technology.51−53 The luciferase activity of the clone was characterized and found to correlate linearly with the cell numbers (Figure 1c). These cells were used to establish the in vivo model and calibrate the experimental parameters such as the cell inoculation number, tumor growth kinetics, and tumor load variability between individual animals (Figure 1d−f, Supplementary Figure S1a,b). The whole body bioluminescence imaging and the analysis of tumor-bearing liver tissues at different time points (e.g., day 8, day 21 and day 26) with fluorescence microscopy led us to the conclusion that the inoculation of 3 × 105 cells results in reproducible liver-confined tumor growth for at least 3 weeks in NSG mice (Figure 1d−f). The tumor colonies are distributed across the entire liver, with more colonies accumulating at the liver periphery. Furthermore, while comparing the metastases distribution in liver sections bearing HCT-116LC tumor nodules stained with Hematoxylin and Eosin (H&E) with the corresponding fluorescence micrograph of mCitrine (Supplementary Figure S1c,d), we confirmed that the formation of the metastases in the liver solely resulted from the HCT-116LC tumor nodules. Delivery Platform Calibration. We considered Adenoassociated virus (AAV) vectors as a classifier circuit delivery and C

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Figure 2. Construction and characterization of AAV miRNA reporters in vitro. (a) Schematic depiction of the reporter constructs. The constitutive bidirectional reporter system is composed of an untargeted fluorescent reporter as an internal control (mCerulean) and a miRNA input-targeted (4xTs) fluorescent reporter that serves as an indicator of miRNA activity (mCherry). The three constructs shown are the control reporter (above) lacking miRNA targets in the 3′-UTR of mCherry, and two reporters of specific miRNA activity (below). AAV2 ITRs (inverted terminal repeats) flank the genetic constructs for AAV packaging. CMVBI, CMVBI-T7 and CMVBI-T122 stand for the plasmids encoding ITR-flanked control, miR-7, and miR-122 reporter constructs, respectively. AAV8-C, AAV6-C, AAV6-T7 and AAV8-T122 refer to the reporters packaged in AAV virions, where the serotype is in the name, “C” stands for control and T7 and T122 refer to miR-7 and miR-122 reporters, respectively. (b, c) Bar charts show the mean fluorescence intensity of mCherry normalized to mCerulean (defined as mCherry fluorescence R.U.) obtained from a triplicate transient transfection in HCT-116 cells of various plasmid-based reporters, as indicated. Yellow bars describe experiments where miR-7 and miR-122 mimics (b) or miR-7 and miR-122 LNA inhibitors (c) are cotransfected. Purple bars describe experiments in which negative control mimics (b) or negative control inhibitors (c) are cotransfected. (d) Flow cytometry scatter plots of HCT-116 cells transfected with various plasmid-based reporters, showing the gating of positive and negative cells and the correlation between mCerulean and mCherry expression. These are shown individually for the indicated constructs (left), and as an overlap (right). (e) The data obtained with AAV-packaged reporters in vitro. Bar chart (top) shows the mean fluorescence intensity of mCherry normalized to mCerulean obtained from a triplicate measurement 3 days post infection with the AAV-packaged reporters in HCT-116LC cells. Corresponding fluorescence micrographs (middle) and flow cytometry plots (bottom) are also shown. Scale bars represent 100 μm.

packaging capacity is limited to 4.4 kb, it should still be able to accommodate classifier circuits with a moderate number of inputs that could suffice for specific cell targeting. Therefore, we

transfer platform. These vectors have been shown to penetrate various tissues efficiently due to their small size,54−57 a feature particularly advantageous for our tumor model. While the D

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ACS Synthetic Biology screened in vitro and in vivo different AAV serotypes expressing constitutive green fluorescent protein (GFP) for their ability to transduce in vivo both the tumor and the healthy cells of interest (hepatocytes in this case) with relatively high efficiency. The broad tropism would allow us to ascertain that the circuit payload, rather than capsid tropism, is the cause of tumorspecific gene activation. AAV6 transduces the HCT-116 cells in vitro with the highest efficiency (Supplementary Figure S2a,b). In vivo, the same panel of serotypes was delivered systemically to mice inoculated with HCT-116LC tumors while varying such parameters as dosage, number of injections, and tumor load at the time of virus injection (Supplementary Figure S2c− g). The data pointed to AAV6 and AAV8 as the most promising candidates due to efficient transduction of both the liver and the tumor in vivo as judged by GFP expression, and helped us to determine the optimal delivery regimen that maximized the transduction efficiency of the tumor cells. In particular, it became clear that one needs to circumvent exponential dilution of this nonreplicating vector resulting from successive doubling of initially transduced tumor cells. Two consecutive injections showed higher overall percentage of infected tumor cells and higher transgene expression in positive cells, suggesting this as a preferred injection regimen (Supplementary Figure S2f). We note the AAV8 transduction efficiency of HCT-116LC is very low in vitro and high in vivo, emphasizing the importance of in vivo screening prior to any decision-making. Identification of Selective miRNA. Following the establishment of the tumor model and the delivery platform parameters, the next step in the translational workflow is to determine the candidate miRNA inputs for cell classifier circuits. Ample data are available pertaining to miRNA expression in normal tissues; however, these data do not always reflect absolute expression, which is the best approximation of a miRNA activity in the absence of direct activity measurements. On the other hand, data on miRNA expression in tumor cell lines is often absent. To create a consistent data set, we opted for in-house miRNA profiling in healthy tissues, HCT-116LC cells, a number of other colorectal cancer cell lines, and in a liver cancer cell line Huh-7, using next-generation sequencing (NGS) on the Illumina platform. In order to obtain reliable absolute expression data of different miRNA we used a spike-in sample of almost 1000 known miRNAs to correct for the ligation bias in the sample preparation protocol, which can result in up to 4 orders of magnitude discrepancy between the counts obtained from miRNAs of identical concentration.58 Even with this correction, we estimate that the confidence interval can be reduced at best to about 10-fold range, that is, a given value can give an estimation of an absolute expression within a 3-fold range. The results (Supplementary Spreadsheet S1 and Supplementary Figure S3) confirmed the high and specific expression of miR122 in the liver15,59,60 and pointed, among other alternatives, to miR-7 as an attractive candidate with high expression in HCT116 cells but low expression in the liver and a number of other tissues. Thus, we chose miR-122 and miR-7 for experimental validation in the animal tumor model. The Construction and Testing of AAV Bidirectional miRNA Activity Reporters. We have previously shown that miRNA activity could be measured in vitro with a tetracycline response element (TRE)-based bidirectional reporter,43 as it displayed excellent correlation between the expression levels of both reporter genes. Because we needed to validate miRNA activity in vivo, we had to design a reporter that did not rely on

transactivation and that could be accommodated in a single DNA construct whose size would permit its packaging in an AAV vector. To this end we designed a reporter based on a strong constitutive CMV enhancer inserted between two minimal CMV promoters. These promoters control the expression of the internal reference fluorescent protein mCerulean and of the reporter fluorescent protein mCherry. mCherry is furnished with four identical tandem repeats of fully complementary targets for the miRNA input of interest in its 3′-UTR (Figure 2a), and therefore its expression is downregulated by this miRNA in a concentration-dependent manner. To ensure that the construct is small enough to be packaged in an AAV vector for in vivo delivery, we did not use expression-boosting genetic elements such as Woodchuck Hepatitis Virus post-transcriptional regulatory elements (WPRE). To characterize and validate in vivo the miRNA candidates selected based on NGS profiling data, we constructed bidirectional reporters as described above to respond to miR7 and miR-122, resulting in the constructs CMVBI-T7 and CMVBI-T122, respectively. We used a control construct without any miRNA targets in the 3′-UTR of mCherry, CMVBI, in order to determine intrinsic mCherry expression (Figure 2a). We first tested the DNA plasmids containing the ITR-flanked reporter genomes in HCT-116 cells in vitro with transient transfection, using synthetic miRNA mimics and Locked Nucleic Acid (LNA)-based miRNA inhibitors to validate reporter specificity and the anticipated miR-7 and miR-122 activity in these cells (Figure 2b,c). We found that miR-7 reporter was downregulated in HCT-116 cells, even though the endogenous levels are not saturating as evidenced by further knockdown in the presence of the miR-7 mimic. MiR-122 elicited only modest mCherry reduction in the cells and this effect appeared nonspecific due to the lack of mCherry upregulation in the presence of miR-122 LNA inhibitor. The lack of specificity is further supported by the qualitative difference in the flow cytometry scatter plots between miR-7 and miR-122: miR-7 reporter data show a pattern typical of genuine miRNA down-regulation41 while the miR-122 reporter data are shifted downward by a constant factor of 2 (Figure 2d). Once we have confirmed differential expression of miR-7 and miR-122 in HCT-116 cells, we proceeded to package their corresponding reporters in AAV particles. Based on the serotype evaluation data (Supplementary Figure S2), we chose AAV6 as a serotype to package the tumor-high miR-7 (AAV6-T7) maker reporter and AAV8 to package liver-high miR-122 reporter (AAV8-T122). This was expected to result in the high in vivo transduction efficiency in the cell type positive for the respective miRNA marker while still enabling the infection of cells supposedly negative for this marker. A bidirectional control reporter without targets was packaged in both AAV6 and AAV8 serotypes. The viruses were tested in vitro in HCT-116LC cells and showed the expected behavior, namely, knockdown of the miR-7 reporter and only minor change in expression of miR-122 reporter. We also observed that the magnitude of mCherry knockdown is much higher with AAV-packaged than plasmid DNA-based reporters (Figure 2e, compare to Figure 2d). The difference in transduction efficiency observed in HCT-116LC cells between AAV6- and AAV8-packaged control reporters is consistent with the data obtained with constitutive GFP reporters (Supplementary Figure S2). E

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Figure 3. Characterization of the miRNA marker activity in vivo. (a) Schematic representation of the in vivo experiment for miRNA marker activity measurement. See main text for experimental groups description. (b) Normalized mCherry expression obtained with various experimental groups (indicated on the X axis) in the liver fraction. Each dot represents the quantitative flow cytometry-based mCherry readout normalized to mCerulean, corresponding to individual animals and resulting from averaging a technical duplicate of dissociated hepatocytes. Representative flow cytometry scatter plots are shown below. (c) Normalized mCherry expression obtained with various experimental groups (indicated on the X axis) in the tumor fraction. Each dot represents the quantitative flow cytometry-based mCherry readout normalized to mCerulean, corresponding to individual animals and resulting from averaging a technical duplicate of dissociated tumor cells. Representative flow cytometry scatter plots are shown below. (d) The fluorescence micrographs of tumor-free fresh liver tissue. Images were taken with 4× objective, 600 ms exposure time for mCerulean and mCherry for AAV6-C and AAV6-T7 groups; 100 ms exposure time for mCerulean and mCherry for AAV8-C and AAV6-T122 groups, and 100 ms exposure time for mCitrine for all the fresh liver tissues. (e) The fluorescence micrographs of tumor nodules isolated from the liver. For all the groups AAV6C, AAV6-T7, AAV8-C and AAV6-T122: Images were taken with 10× objective using 1000 ms exposure for mCerulean, 600 ms exposure for mCherry, and 30 ms exposure time for mCitrine. Scale bars represent 100 μm.

Bidirectional Reporters In Vivo. To assess reporter specificity in the in vivo model, we delivered the viruses systemically via tail vein injection (IV) to tumor-bearing NSG mice. After the initial tumor inoculation, mice were assigned to five groups using stratified randomization with in vivo whole body luminescent signal intensity as a covariate to ensure similar tumor load distribution between groups. The five

groups were administered, respectively, with sham injection (PBS), control reporter vectors AAV6-C and AAV8-C, and the miRNA reporters AAV6-T7 and AAV8-T122 (n = 4 per group). As described above (Supplementary Figure S2f,g), two consecutive injections 7 and 9 days post tumor inoculation were used to increase the percentage of tumor cells expressing fluorescence above detection threshold and the absolute F

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Figure 4. Characterization of the cytotoxic HSV-TK/GCV output activity in vivo. (a) Schematic depiction of the HSV-TK delivery construct as an ITR-flanked insert with miR-122 target sites (4xTs). AAV6-HSVTK-T122 refers to the construct packaged in AAV6 vectors. EF1Ashort stands for a synthetic size optimized promoter based on native Ef1α promoter architecture, and HSV-TK stands for the Herpes Simplex Virus Thymidine Kinase. (b) Schematic representation of the in vivo experiment for the determination of HSV-TK output effect in vivo. See main text for experimental groups description. (c) Tumor growth curves based on in vivo luciferase activity measured by whole body bioluminescence imaging after inoculation of 105 HCT-116LC cells. Each circle refers to the average value of the AAV6-HSVTK-T122 + GCV and of the AAV6-HSVTK-T122 + saline group or individual data points of the PBS group as indicated in the legend. Each trend line represents the moving average of period 3 for all groups. (d) Fluorescence micrographs of liver tissues obtained from tumor-inoculated animals treated with cytotoxic construct with and without GCV, or with sham treatment (PBS). Images were taken with 4× objective and 100 ms exposure for mCitrine and mCherry. (e) Fluorescence micrographs of fresh brain tissue. Images were taken with 4× objective and 600 ms exposure for mCherry. Scale bars correspond to 100 μm.

reporter expression in these cells at termination. Measurements and quantitative analysis of mCherry and mCerulean signals were performed at end point 15 days post tumor inoculation in healthy tissues and tumors. To evaluate reporter knockdown at the single cell level in the liver and in the tumor, tumor-bearing liver lobes were collected and digested briefly using collagenase IV/Dispase II to dissociate the hepatocytes (“liver fraction”) while leaving the tumor nodules relatively intact. Next, the tumor nodules were collected and further digested into single cells (“tumor fraction”) using Accutase (Figure 3a). The two fractions were analyzed by flow cytometry (Supplementary Figure S4) to evaluate the fluorescent protein expression at a single cell level. In order to derive quantitative data, the mCherry signal was normalized by the internal mCerulean control. As expected, this normalization proved to be extremely valuable as it allowed compensating for the large variability in transduction efficiency between different viral serotypes, tissues and cell types, and animals. The data show that the expression of mCherry from

AAV8-T122 compared to AAV8-C control was down-regulated more than 500-fold in the liver, with virtually zero cells in this fraction expressing detectable mCherry levels (Figure 3b). This is consistent with the published data,46,61 whereas the presence of the internal control mCerulean further increased the confidence in this conclusion. Interestingly, the images of tumor-bearing liver obtained from the mouse injected with AAV8-T122 show tumor nodules expressing mCherry on Cherry-negative background, providing perhaps the first direct evidence that miRNA regulation can specifically target gene expression to the tumor site despite delivery via systemic route (Supplementary Figure S5). Quantitatively, and as confirmed by image examination, the miR-122 reporter also showed 7-fold downregulation in the tumor cell fraction relative to the control (Figure 3c), much higher than the apparent 2-fold downregulation that had been observed in vitro. The expression of mCherry from AAV6-T7 compared to AAV6-C was downregulated by a factor of about 70 in the tumor cells (Figure 3c), consistent with the in vitro G

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injections of saline solution of 0.9% NaCl), those injected with AAV6-HSVTK-T122 followed by the administration of the prodrug GCV (daily IP injection of 100 mg/kg62) (n = 2 per group) and a control mouse that received sham tail vein injection (PBS) (Figure 4b). Two consecutive injections of the viral vector (4 and 8 days post tumor inoculation) were used. Daily prodrug treatment of each mouse with GCV or control saline was initiated 6 days post tumor inoculation. During the treatment, tumor growth was monitored with bioluminescence imaging every 2 days for a period of 20 days, after which the mice were euthanized for tumor load assessment using fluorescence microscopy. The tumor growth monitoring revealed that tumor regression started a week after commencing prodrug administration and was sustainable for more than a week in the AAV6-HSVTK-T122 + GCV group compared to the AAV6-HSVTK-T122 + saline and PBS groups (Figure 4c). In order to assess tumor load in the liver, we imaged different areas of the liver lobes using fluorescence microscopy. The images revealed almost complete absence of tumor microcolonies (mCitrine-positive nodules) in the liver of the animals in the AAV6-HSVTK-T122 + GCV group. On the other hand, multiple tumor nodules of various sizes were observed in groups treated with either AAV6-HSVTK-T122 + saline or PBS (Figure 4d). The combination of in vivo luminescence and microscopy imaging confirms the intrinsic antitumor efficacy of the HSV-TK/GCV in combination with AAV6 delivery in our animal model. Normal serum AST/ALT data (data not shown) confirmed that the miR-122 protection of liver expression, observed directly with the fluorescent reporter, was also able to efficiently control the cytotoxic output in that organ. Nevertheless, systemic output toxicity was still apparent, as evidenced by 15% weight reduction of GCV-treated animals compared to the control groups. This emphasizes the importance of the multi-input control for output fine-tuning to eradicate toxicity in other organs and widen the therapeutic window. The systemic HSV-TK/GCV cytotoxicity is further supported by the fluorescent images of tissues collected from various organs, in particularly the brain, where we not only detected high expression of mCherry, but also observed altered morphology of the mCherry-expressing cells (Figure 4e).

data. Quite unexpectedly, mCherry in AAV6-T7 was reduced around 6 fold in the liver fraction relative to AAV6-C despite NSG data that did not indicate any miR-7 in this tissue (Figure 3b). In parallel, we collected thinly sliced tissue fragments from the liver, brain, kidney, pancreas, heart, lungs, and muscles. We imaged the reporter outputs in these freshly prepared tissues to assess viral distribution and to see whether the two miRNA of interest are also expressed there (Figure 3d, Supplementary Figure S6a−f). Likewise, we imaged intact tumor nodules isolated from the tumor-bearing liver (Figure 3e). In summary, AAV6-C infected the tumor, liver, pancreas, lungs, and brain; AAV8-C infected the tumor, liver, pancreas, and kidneys. Because the pancreas was infected strongly with both AAV8-C and AAV8-T122, we performed quantitative image analysis to check if there is any residual difference in normalized mCherry expression from the two constructs. Fields of view with relatively uniform background were chosen in the four pancreas images from AAV8-C- and AAV8-T122-infected mice, and the mCherry and mCerulean intensity was integrated after background correction using two different methods. In summary, both methods show that the normalized mCherry expression in AAV8-T122 is about 75% of the expression in AAV8-C control, consistent with the difference observed in vitro (Supplementary Figure S6g); therefore this confirms that the effects observed in the liver with miR-7 reporter, and in the tumor with miR-122 reporter, are genuine downregulation that might result from miRNA exchange between the tumor cells and hepatocytes via exosomes or by cross-talk between the miR-7-like sequences and AAV6-T7 reporter in the liver. To further confirm that the mere presence of a tandem target repeat in the reporters and its absence in the control cannot explain ∼7-fold difference in the reporter readout, we created a construct CMVBI-TFF5, including four repeats of complementary sequence to a synthetic miR-FF5. We tested the DNA plasmids (CMVBI-TFF5 and CMVBI) in HCT-116LC (Supplementary Figure S7) and found, similar to pancreas data, that miR-FF5 reporter was only slightly downregulated in these cells relative to the CMVBI. Evaluation of the Cytotoxic Output HSV-TK/GCV. The therapeutic effect of a classifier relies on the intrinsic antitumor efficacy of its output in vivo, because the classifier only controls output expression in different cell types. To assess the intrinsic efficacy of a candidate output in our experimental conditions, we chose to deliver the cytotoxic HSV-TK gene in an AAV6 construct coexpressing a constitutive fluorescent reporter mCherry (AAV6-HSVTK-T122, Figure 4a). We used an architecture similar to CMVBI, but replaced one of the minimal CMV promoters with an optimized synthetic promoter based on native Ef1α promoter architecture, EF1Ashort (704 bp), to drive the HSV-TK. The EF1Ashort was used due to its potential to drive the output in compact gene circuits. HSV-TK gene was furnished with four repeats of miR-122 targets to protect the animal from acute toxicity46 upon systemic delivery. The reporter mCherry allows tracking infected organs for potential damage related to the expression of HSV-TK combined with GCV treatment. After the initial tumor inoculation, mice were assigned to three groups in a stratified fashion using in vivo whole body luminescent signal intensity as a covariate to ensure similar tumor loads between groups. The groups comprised mice treated with tail vein injection of the cytotoxic AAV6-HSVTKT122 without follow-up prodrug administration (sham daily IP

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DISCUSSION Mammalian synthetic biology has focused so far on developing gene circuit technologies to achieve various tasks, predominantly in the biomedical application area. However, most proof of concept experiments have been done in cell culture. The next step for these technologies is a demonstration in vivo toward clinical translation. This brings many confounding factors that must be taken into account while being outside of the direct focus of most synthetic biology researchers. One research direction in the field is genetic cell classifiers for specific cell targeting, or, more generally, systems that are designed to interact with endogenous intracellular signals. The physiopathology of the clinical disease and of the corresponding animal disease models, in combination with the prospective delivery platforms, play the key role in determining whether the circuits can reach the disease site and interact with the endogenous signals in a way that is sufficient for their intended functionality. In this study we describe and experimentally illustrate a workflow that should precede an in depth preclinical evaluation of gene circuit technologies. In our case, we chose a murine model of metastatic tumor in the liver to demonstrate the H

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group. These experiments are important not only to establish the reference for output efficacy, but also to evaluate both efficacy and toxicity prior to circuit studies. In many gene circuits with therapeutic potential, the circuits control the expression of output products that are known to have an intrinsic therapeutic effect against the disease under investigation. In such cases, the comparison of the circuit treatment should not only be done to the placebo (in which case a positive effect is likely to be detected due to the presence of the output per se), but also to the constitutive or minimally controlled outputs, as we have done using miR-122 targets. The evaluation of the complete circuit at the next stage should focus predominantly on side effects, the width of the therapeutic window, etc. In our case, furnishing the cytotoxic output with a coexpressed fluorescent reporter also allowed direct visualization of the tissues affected by the nonspecific toxicity. This, together with the toxicity apparent from animal weight loss and change in brain cells morphology, form the reference for evaluating whether multi-input circuits indeed result in reduced toxicity in bystander tissues without reducing antitumor effects, as hypothesized.

feasibility of the workflow due to the disease’s high unmet medical need and due to the fact that the liver is an attractive anatomical environment for AAV-based tumor targeting as high blood flow in the liver and porous vessel structure lead to high liver accumulation of the virus. This was shown to be largely the case, and novel and improved AAV capsids are likely to increase tumor infectivity further.56 In parallel, classifiers must be adapted to the animal model under consideration. In most cases, published expression levels of different miRNAs, or even levels measured in house using common profiling methods, do not provide reliable information regarding marker activity in vivo. Therefore, the highest-ranking candidates based on a combination of published and in-house measurements must be tested and confirmed experimentally in vivo using dedicated reporters before being incorporated into circuit design. This was accomplished by engineering a novel AAV-based miRNA activity reporter system that is directly compatible with and comparable to the delivery system envisioned for the fully functional disease-targeting circuits. As our study shows, in order to get meaningful in vivo data toward eventual translation to medical applications, we needed to optimize the experimental procedure of establishing the tumor model and of delivering the viral vectors. This included calibrating the number of inoculated cells, the wait time/tumor load prior to viral injections, the injected viral titer and the number of injections, and the termination end points. This optimization was only possible because of extensive fluorescent labeling of the tumor cells and viral vectors, allowing us to obtain detailed and quantitative spatial data. After optimizing these parameters, we were able to get reproducible quantitative information on the in vivo activity of each miRNA input. These parameters are not universal, and much depends for example on the biology of the tumor cells and the interaction between the cells and their physiological environment. Eventually, experiments with patient-derived tumors will give more clinically predictive data regarding future translation. A noteworthy result that became apparent using in vivo reporters was downregulation of miR-122 reporter in the tumor and of miR-7 reporter in the liver. While miR-7 reporter in the liver might in principle be downregulated nonspecifically by crosstalk with other sequences, the downregulation of miR-122 reporter was not observed in vitro and is therefore a result of tumor residing in the liver and close proximity to hepatocytes rich in miR-122. The close proximity of tumor and hepatocytes suggests that exosome-mediated transfer63 of miRNA could have contributed to the transfer of miRNA-7 and miRNA-122 resulting in downregulation observed in vivo. In addition, the quantitative measurement of in vivo marker activity, namely 500-fold downregulation by miR-122 in the liver and 70-fold downregulation by miR-7 in the tumor, provides valuable information for these inputs’ incorporation in cell classifiers. This observation emphasizes further the importance of in vivo miRNA quantification using, e.g., the bidirectional reporters described here, without which important information on spatial activity of miRNA under a specific pathophysiological condition would be lost. The apparent exchange of the miRNA also suggests that the sensor sensitivity should be adjusted to reduce the contribution of the foreign input while retaining the sensitivity to the desired input. Lastly, we confirmed the intrinsic antitumor efficacy of HSVTK/GCV combination in our specific conditions as suggested by a decrease in tumor growth rate, leading to the cessation of tumor growth in the treated group compared to the untreated

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METHODS Plasmids Construction. Plasmids were constructed using standard cloning techniques. One Shot Stbl3 Chemically Competent E. coli (Life Technologies, C7373−03) was used as the cloning strain, cultured in LB Broth Miller Difco (BD) supplemented with appropriate antibiotics (Ampicillin, 100 μg/ mL, Chloramphenicol, 25 μg/mL, Kanamycin, 50 μg/mL). All enzymes were purchased from New England Biolabs (NEB). Phusion High-Fidelity DNA Polymerase (NEB) was used for fragment amplification. Single stranded oligonucleotides used as primers or for self-annealing were provided by Microsynth or Sigma-Aldrich. Digestion products and PCR fragments were purified by either using Gen Elute PCR Clean Up Kit (SigmaAldrich) or running an agarose gel electrophoresis with subsequent gel extraction using the GenElute Gel Extraction Kit (Sigma-Aldrich). All ligations were performed using T4 DNA Ligase (NEB) or Quick Ligation Kit (NEB), with individually adjusted incubation time and temperature. Ligation product was transformed into Stbl3 and plated on LB Agar plates with appropriate antibiotics. After plasmid DNA purification of individual clones using GenElute Plasmid Miniprep Kit (Sigma-Aldrich), the sequencing was done by Microsynth. A short cloning procedure of each construct used in this work is as follows. CMVBI: CMVbi bidirectional reporter (pMD26) was constructed as the following: pBH007443 was cut with BamHI and MluI blunted and religated. The CMV promoter/enhancer element was PCR-amplified from pJS114 with primers PR2912/14 and cloned into the EcoRI/MscI sites of the religated pBH0074. This construct was SspI-digested, blunted and cut with SalI. The insert was cloned into the SalI and NruI (blunted) sites of pMD6 ITRs. The second CMV was PCR amplified from pZ088 with primers PR3298/PR2399 and cloned into the EcoRI-SpeI sites. While checking the integrity of the ITRs of this vector we noticed an 11 bp deletion in the 5′ITR. We designed oligos PR3070/71 to substitute the deletion with the correct sequence by insertion into the PciI and MluI sites. CMVBI-T7 (pMD31): pMD26 was digested using BamHI and SalI and was ligated with annealed and phosphorylated oligos PR2926 and PR2927, coding for 4 repeats of miR-7 target. CMVBI-T122 (pMD30): pMD26 was digested using BamHI and SalI and was ligated with annealed and I

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Plasmids were purified from 100 mL cultures of E. coli Stbl3 grown overnight at 37 °C at 200 rpm in LB Broth Miller Difco (BD) supplemented with appropriate antibiotic using HiPure Plasmid Filter Maxi Kit (Invitrogen, K210014). After plasmid purification an additional purification step was performed using Endotoxin Removal Kit (Norgen Biotek Corporation). DNA amounts were quantified using Nanodrop (ND-2000) and integrity was verified by agarose gel electrophoresis. The purified plasmids were mixed as required and diluted with 50 μL Opti-MEM I Reduced Serum (Gibco, Life technologies Cat # 31985-962) per sample for 24 well plates, respectively. If needed, microRNA mimics and LNA-inhibitors were added to the plasmid mix. Mimics were purchased from Dharmacon (Mim-7, C-300547-05, Mim-122, C-300591-05, Mim-Neg.Ctrl., CN-001000-01-05), LNA inhibitors form Exiqon (LNA-7 4100814-101, LNA-122 0.426674-00, LNA-Neg.Ctrl. 19902000). Transfections were performed as suggested by the manufacturer. Construction of Luciferase and mCitrine-Expressing Cell Line. 106 HCT-116 cells were seeded in a 6-well plate and transfected with 2 μg DNA (500 ng hAAVS1 1L TALEN, 500 ng hAAVS1 1R TALEN, 1 μg pIK014) using Lipofectamine 2000. Transformed cells were selected for 1 week with puromycin (Invivogen, 16A29-MM) at 0.8 μg/mL. Positive clones were isolated and cultured in 96 wells, then expanded from 24-well to 6-well plates for cell sorting of mCitrine+ cells using a BD FACS Aria III. Cell pellets were analyzed using colony PCR to validate the presence of the construct. Functional luciferase activity was measured as following; different numbers of HCT-116LC cells (20 000 down to 313 cells) were seeded into a 96 well black plate (Greiner Zellkultur Microplatten 96 Well, 7.655 090). After 5 h medium was supplanted with 100 μL Luciferin 150 μg/mL (Beetle Luciferin, Promega, AG E1605) and cells where incubated at RT for 15 min. Bioluminescence was measured for 2 min using the PhotonIMAGER RT (Biospace Laboratories). Production, Purification and Quantification of SingleStranded AAV Vectors. Single-stranded (ss) AAV vectors were produced and purified as previously described.67,68 Briefly, human embryonic kidney cells (HEK293) expressing the simian virus69 large T-antigen (293T)70,71 were cotransfected with polyethylenimine (PEI)-mediated AAV vector plasmids (providing the to-be packaged AAV vector genome), AAV helper plasmids (providing the AAV serotype 2 rep proteins and the cap proteins of the AAV serotype of interest) and adenovirus (AV) helper plasmids pBS-E2A-VA-E472 (providing the AV helper functions) in a 1:1:1 molar ratio. 96 to 120 h post transfection HEK293T cells were collected and separated from their supernatant by low-speed centrifugation (15 min at 1500g/4 °C). AAV vectors released into the supernatant were PEG-precipitated overnight at 4 °C by adding PEG 8000 solution (final: 8% v/v) and NaCl (final: 0.5 M). PEGprecipitation was completed by low-speed centrifugation (60 min at 3488g/4 °C). Cleared supernatant was discarded and the pelleted AAV vectors resuspended in AAV resuspension buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.5). HEK293T cells were resuspended in AAV resuspension buffer and lysed by Bertin’s Minilys Homogenizer in combination with 7 mL soft tissue homogenizing CK14 tubes (two 1 min cycles at 5000 rpm/RT, intermitted by >4 min cooling at −20 °C). The crude cell lysate was treated with the BitNuclease endonuclease (75 U/mL, 30 to 90 min at 37 °C) and cleared by centrifugation (10 min at 17 000g/4 °C). The PEG-pelleted AAV vectors were

phosphorylated oligos PR2916/PR2917, coding for 4 repeats of miR-122 target. CMVBI-TFF5 (pMD64): pMD26 was digested using BamHI and SalI and ligated with annealed and phosphorylated oligos PR3511/PR3512, coding for 4 repeats of synthetic miRNA-FF5 targets. Cerulean-EF1Ashort-CMVmCherry (pMD34): pMD26 was digested with EcoRI and SpeI and ligated with PCR-amplified EF1A short promoter with primers PR3476/PR3477 from pJD19. HSVTK-EF1AshortCMV-mCherry (pMD41): pMD34 was digested with EcoRI and HindIII and ligated with HSVTK PCR-amplified fragment with primers PR3607/PR3608 from pBA696. T122-HSVTKEF1Ashort-CMV-mCherry (pMD43): pMD41 was digested with HindIII and XbaI and ligated with phosphorylated and annealed oligos PR3499/P3500 to add the 4 repeats of miR122 target site. Ef1a-mCitrine-T2A-luciferase2 (pIK013): Gibson assembly was performed with a digested fragment of p0001 with NsiI and MfeI (for the backbone), and with PCR amplification with primers (PR1607/PR1608) for EF1apromoter from pKH025,64 with primers for mCitrine (PR1611/PR1612) from pKH025 64 and with primers PR1609/PR1610 for luciferase-2 from the pGL4.13 reporter vector. During PCR amplification, T2A was introduced partly at the 3′ end of luciferase-2 and 5′ end of mCitrine sequences by the respective primers. AAVS1 SA-2A-puro-pA donor Backbone (pIK006): pIK004 (#22075, addgene) was digested with SalI and was religated with annealed oligos PR1233/PR1232 to create a multiple cloning site. AAVS1 SA-2A-puro-pA donor plasmid (pIK007): The plasmid pIK005 (ins-EF1a-mCitrineT2A-luciferase-polyA-ins) was cut with PspXI and SalI and the AAVS1 SA-2A-puro-pA donor Backbone (pIK006) was cut with PspXI, the bands were ligated. AAVS1-T2A-puro-polyAins-Ef1a-mCitrine-T2A-luciferase2-polyA-ins-AAVS1 (pIK014): pIK013 was digested with NsiI and PspXI and cloned into pIK007 the AAVS1 SA-2A puro pA donor plasmid (#22075, addgene), where a multiple cloning site was inserted between polyA and AAVS1 arm, applying standard digestionligation cloning strategy. Briefly, the donor plasmid and plasmid pIK013 were digested with NsiI and PspXI and the bands were ligated. AAVS1 SA-2A-puro-pA donor (pIK004): Commercial plasmid from (#22075, addgene), described earlier.65 hAAVS1 1L TALEN (pIK011): plasmid # 35431 (Addgene).53 hAAVS1 1R TALEN (pIK012): plasmid # 35432 (Addgene).53 AAVCK(0.4)GW (pMD6): plasmid # 27226 (Addgene).66 Ef1αmCitrine (pKH025): described in Prochazka et al.64 pGL4.13: Commercial plasmid E6681 (Promega). pBA696 (HSVTK Gene): plasmid # 21911 (Addgene). The primers are listed in Supplementary Table S1. Cell Culture and Transfection. HCT-116 cells were obtained from Deutsche Sammlung von Microorganismen and Zellkulturen (DSMZ), DSMZ No ACC-581, and cultured at 37 °C, 5% CO2 in DMEM, high glucose, GlutaMAX (Life Technologies, 31966-021), supplemented with 10% FBS (Sigma-Aldrich, F9665 or Life Technologies, 10270106) and 1% Penicillin/Streptomycin Solution (Sigma-Aldrich, P4333). Cells were passaged every 3−4 days using StemPro Accutase (Life Technologies, A11105-01). Transfection was performed using Lipofectamine 2000 transfection reagent (Life Technologies, 11668-019) in uncoated 24-well plates (Thermo Scientific, 142475). For transfections in 24 well plates, HCT116 cells were seeded 10 h before transfection at a density of 125 000 cells/well in 500 μL of complete medium. The medium was replaced before transfection with fresh medium. Transfections were performed at 60−80% cell confluence. J

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ACS Synthetic Biology combined with the cleared lysate and subjected to discontinuous density iodixanol (OptiPrep, Axis-Shield) gradient (isopycnic) ultracentrifugation (2 h 15 min at 365 929g/15 °C). Subsequently, the iodixanol was removed from the AAV vector containing fraction by three rounds of diafiltration (ultrafiltration) using Vivaspin 20 ultrafiltration devices (100 000 MWCO, PES membrane, Sartorius) and 1× phosphate buffered saline (PBS) supplemented with 1 mM MgCl2 and 2.5 mM KCl according to the manufacturer’s instructions. The AAV vectors were stored aliquoted at −80 °C. Encapsidated viral vector genomes (vg) were quantified using the Qubit 3.0 fluorometer in combination with the Qubit dsDNA HS Assay Kit (both Life Technologies). Briefly, 5 μL of undiluted (or 1:10 diluted) AAV vectors were prepared in duplicate. One sample was heat-denatured (5 min at 95 °C) and the untreated and heat-denatured samples were quantified according to the manufacturer’s instructions. Intraviral (encapsidated) vg/mL were calculated by subtracting the extraviral (nonencapsidated; untreated sample) from the total intra- and extraviral (encapsidated and nonencapsidated; heatdenatured sample). In Vitro Infection. HCT-116 or HCT-116LC cells were seeded in 12-well plates (Multiwell Culture Plate 12 well NUNC, NC-150628) and infected at a density of 250 000 cells per well. For each in vitro infection, the numbers of virus particles (viral genomes/well) is described in Supplementary Table S2. Each infection was done in triplicate. After 72 h, infectivity was analyzed using flow cytometry and fluorescence microscopy. Cell Preparation for In Vivo Inoculation. HCT-116LC cells were cultured and passaged until 70−80% confluence in 10 cm Petri dishes. HCT-116LC single cell suspension was obtained by removing the growth medium, washing with 10 mL PBS (twice), and dissociating the cells with 2 mL per dish of StemPro Accutase (Life technologies, A11105−01) for 10−13 min at 37 °C, gentle cell suspension by pipetting, and subsequent cell filtering using a 40 μm filter. The cell suspension was supplemented with 8 mL PBS and centrifuged at 498 rpm at 4 °C for 9 min. The cell pellet was washed with PBS and centrifuged at 498 rpm at 4 °C for 6 min two more times. The remaining cell pellet was suspended in PBS for manual counting of live cells using Neubauer chamber and trypan blue. At least four independent counts were taken per cell suspension and the average value was used to determine the number of cells to be injected. Cell suspension was inspected visually under the microscope to make sure that there are no cell clumps. At the end the volume was adjusted with PBS to about 6 × 106 cells/mL. The cell suspension was kept on ice for the duration of the surgeries. We note that both the presence of cell clumps and the presence of residual Accutase or other celldissociation reagents is toxic and potentially life-threatening to the animals. Animal Surgeries for Cell Inoculation. 6−8 weeks old female NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ, Charles River Laboratories, Sulzfeld, Germany) were housed in specific pathogen free (SPF) facility. All animal procedures were performed in accordance with the Swiss federal law and institutional guidelines of Eidgenössische Technische Hochschule (ETH) Zurich, and approved by the Animal ethics committee of canton Basel-Stadt (approval numbers 2673/ 24596 and 2673/28204). Experimental surgeries for establishment of colon cancer metastases in the liver were performed under isoflurane anesthesia based on the described procedure,47

as follows. A skin incision of 1−1.5 cm is made in the left lateral flank. The peritoneal cavity is entered and the spleen is exposed and exteriorized. 50 μl of tumor cell suspension containing the required number of cells in PBS are injected into the spleen parenchyma using Hamilton syringe. After 10 min, which provides sufficient time for the majority of cells to reach the liver for colonization, the major splenic vasculature is tightly ligated with 6−0 silk sutures and the spleen is removed to prevent intrasplenic tumor growth. The peritoneal wall is closed with a simple running stitch using 6−0 PDS II suture, and skin is closed with 7 mm wound clips. The tumor growth in NSG mice is monitored by bioluminescence imaging two to three times per week (PhotonIMAGER RT, Biospace Lab) under isoflurane anesthesia for 10 min post I.P (intra peritoneal) injection of 75 mg/kg luciferin (Beetle Luciferin, Promega, AG E1605). The tumor load was inferred by bioluminescent signals Ph/s/sr observed in the liver area. In Vivo AAV Fluorescent Reporter Delivery Regimen. For the in vivo fluorescent reporter experiments (Figure 3 and Supplementary Figures S4−S6), the mice were assigned to groups at day 7 when tumor reached a signal of about 106 Ph/ s/sr. Animals were grouped using stratified approach with the tumor bioluminescent signal as a covariate to ensure similar initial tumor load distribution between groups (n = 4). The animals were injected in the tail vein with the AAV viral batches (Supplementary Table S2). 3.75 × 1012 viral genome particles were diluted in PBS and delivered in two consecutive injections (7 days and 9 days post surgery) as described in Supplementary Table S2. Mice were euthanized 6 days after the first injection. For the in vivo experiment with GFP positive viruses (AAVGFP) and HCT-116LC tumor (Supplementary Figure S2), mice were injected via tail vein with PBS or one of the AAVGFP as described in Supplementary Table S2 6 days post surgery when the tumor load reached on average 106 Ph/s/sr. Each virus construct was diluted in PBS and 1 × 1012 viral genome particles were injected with a single (day 6) or double injection (day 6 and 9) as described in Supplementary Table S2. Animals were euthanized 6 days post first virus injection. In Vivo AAV Cytotoxic Agent Delivery Regimen. To study the cytotoxic output efficacy with the AAV6-HSVTKT122 construct, 4 days post surgery the mice were stratified based on bioluminescent intensity and randomly assigned into two groups (GCV and saline) such that the tumor load is comparable between groups (n = 2) to receive tail vein injection of the AAV6-HSVTK-T122 construct or PBS (Supplementary Table S2). 4 × 1012 viral genome particles were diluted in PBS and delivered via two consecutive injections (at day 4 and day 8 post surgery) as described in Supplementary Table S2. Prodrug treatment was administrated daily via intraperitoneal injection of filtered 100 mg/kg GCV solution (10 mg/mL saline solution, 12CB13-MM sud-gcv Ganciclovir, InvivoGen) for the GCV group; saline was administrated daily intraperitoneally for the “saline” group. Tumor growth was monitored with bioluminescent imaging every two or 3 days (see above). Mice were euthanized 16 days after the first virus injection. Tissue Processing and Analysis. To remove red blood cells prior to flow cytometry analysis and tissue imaging, we performed an intracardiac perfusion with 50 mL of a solution containing 25 units/mL heparin (Sigma-Aldrich, H3149−100 KU) in 1× HBSS (Life Technologies, 14175−053). After perfusion, several fresh tissues (liver, brain, pancreas, kidney, heart, muscles, lungs) were placed in PBS on ice for K

DOI: 10.1021/acssynbio.7b00303 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology

magnifications of all experiments are indicated in the figure legends or in the panels. Flow Cytometry. All cell culture samples were analyzed 48 h after transfection or 72 h post infection, using a BD LSR Fortessa cell analyzer. The medium was removed and cells were incubated with 150/50 μL phenol-red free Trypsin (0.5% Trypsin-EDTA (Gibco, Life Technologies, 15400-054) 1:2 diluted with PBS (Life Technologies, 10010-56). Dispersed cells were transferred to FACS tubes (Life Systems Design, 021412-000) and kept on ice. For the analysis of the liver and tumor fractions (see above for dissociation methods), each sample was twice run on a BD LSR Fortessa cell analyzer. For the detection of the different CMVBI fluorescent reporters, we used reporter-specific combinations of excitation lasers and emission filters. For mCherry we used a 561 nm excitation laser, 600 nm long-pass filter, and 610/20-emission filter. For mCitrine we used 488 nm laser, 505 nm long-pass filter, and 542/27 nm emission filter. For mCerulean we used a 445 nm laser and a 473/10 nm emission filter. For GFP, we used a 488 nm excitation laser, 505 nm long-pass filter, and 530/30 nm emission filters. In the experiments with GFP expressing viruses and HCT-116LC tumor (Supplementary Figure S2): for mCitrine we used a 488 nm excitation laser, 525 nm longpass filter and 542/27 nm emission filter. For the GFP experiment with HCT-116LC cells we used 488 nm excitation laser, 495 nm long-pass filter and 510/20 nm emission filter. PMTs were adjusted using standard fluorescent beads before and after each measurement in order to preserve constant device performance. Data Analysis. All flow cytometry data were analyzed using FlowJo software. For the detection of the different CMVbi fluorescent reporters, a compensation of minor cross talk of mCitrine 488−542/27 nm channel into the mCerulean 445− 473/10 nm channel (