Patent Application
20180216112
The present invention concerns a process for inducing resistance to diphtheria toxin in human cells, products and uses thereof.
Stable transduction, i.e. permanent integration of exogenous genetic material in the cellular genome, is widely employed in basic and applied biomedical research.
In the case of human cells, this is typically achieved by transduction with an expression vector encoding resistance proteins for cytotoxic antibiotics like G418, puromycin, hygromycin and others. Pharmacologic selection then yields a cell population constitutively expressing the resistance marker, plus additional genetic elements of interest.
However, a major limitation of currently available selectable markers lies, in fact, in the impossibility to apply any selection process in vivo: the drugs used for selection are toxic for all eukaryotic cells and therefore would be lethal if administered to a mouse hosting human cells, e.g. a human tumor xenograft.
This restriction is not particularly relevant for cell lines, which can be transduced and selected in vitro and then implanted or injected into immunocompromised mice.
It is instead a crucial drawback for patient-derived cancer xenografts (PDXs), which are directly propagated from human tumour tissue into immunocompromised mice and for which no procedures for in vivo stable transduction have been found to work efficiently, with the exception of constructs that directly provide a selective advantage during outgrowth.
Such methodology is not suitable for basic tumor marking or for therapeutic proof-of-concept studies in which expression of the sequence of interest would instead be detrimental to cancer cells.
Ex vivo transduction strategies, by which cells are separated from explanted tissues, transduced and reimplanted, have also been described. However, the complexity and low efficiency of this approach pose severe limits to their practical application. Moreover, in vitro culture of PDX-derived cancer cells has been shown to deeply and irreversibly alter their gene expression profile, and therefore their functional state.
Therefore, it is felt the need for the identification of new selectable markers usable in in vivo selection of human cells for marker studies or for therapeutic target validation.
The object of the present invention is to provide a new selectable marker conferring resistance to DT usable either in in vitro or in in vivo selection of human cells, wherein the in vivo selection is carried out in a rodent xenografted with human cells.
According to the invention, the above object is achieved thanks to the method specified in the ensuing claims, which are understood as forming an integral part of the present description.
In an embodiment, the instant disclosure discloses a process for inducing resistance to diphtheria toxin in a human cell, wherein the process is carried out in vitro and/or in vivo in a rodent xenografted with a human cell, the process comprising the following steps:
a) providing at least one expression vector containing at least one nucleotide sequence targeting by RNA interference the DPH2 gene transcript of the human cell;
c) transducing the at least one vector into the human cell;
d) allowing transcription in the human cell, from the at least one expression vector, of the at least one nucleotide sequence targeting by RNA interference the DPH2 gene transcript;
e) obtaining silencing of the DPH2 gene in the human cell, whereby the human cell is resistant to diphtheria toxin.
In a further embodiment, the instant description discloses a transgenic diphtheria toxin resistant human cell comprising at least one expression vector containing at least one nucleotide sequence targeting by RNA interference the human DPH2 gene transcript, wherein transcription of the at least one expression vector silences the DPH2 gene in the human cell.
According to a still further embodiment, the instant description concerns use of an expression vector containing at least one nucleotide sequence targeting by RNA interference the DPH2 gene transcript of a human cell for inducing resistance to diphtheria toxin in a human cell, wherein the vector is used in vitro and/or in vivo in a rodent xenografted with a human cell, wherein the transcript of the expression vector is permanently transduced in the human cell, whereby the permanently transduced human cell survives upon diphtheria toxin treatment. In an further embodiment, the expression vector contains at least one additional nucleotide sequence, whereby the permanently transduced human cell surviving upon diphtheria toxin treatment contains the at least one additional nucleotide sequence.
The invention will now be described, by way of example only, with reference to the enclosed figures of drawing, wherein:
The invention will now be described in detail, by way of non limiting example, with reference to a shRNAmir sequence able to silence DPH2 gene in human cells in order to obtain human cells which are resistant to diphtheria toxin.
It is clear that the scope of this description is in no way limited to the specifically disclosed shRNAmir sequence, being possible making use of nucleotide sequences targeting by RNA interference the DPH2 gene transcript of human cells incorporated in double stranded RNA, short hairpin RNA or microRNA.
In the following description, numerous specific details are given to provide a thorough understanding of embodiments. The embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.
Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
The present invention concerns a process for inducing resistance to diphtheria toxin in human cells, the process being usable either in in vitro or in in vivo selection of human cells, wherein the in vivo selection is carried out in a rodent xenografted with human cells.
The present description concerns a process for inducing resistance to diphtheria toxin in a human cell, wherein the process is carried out in vitro and/or in vivo in a rodent, preferably a mouse, xenografted with a human cell, the process comprising the following steps:
a) providing at least one expression vector containing at least one nucleotide sequence targeting by RNA interference the DPH2 gene transcript of the human cell;
c) transducing the at least one vector into the human cell;
d) allowing transcription in the human cell, from the at least one expression vector, of the at least one nucleotide sequence targeting by RNA interference the DPH2 gene transcript;
e) obtaining silencing of the DPH2 gene in the human cell, whereby the human cell is resistant to diphtheria toxin.
In a preferred embodiment, the transcript of the at least one expression vector is permanently transduced into the human cell.
In a still preferred embodiment, the at least one nucleotide sequence targeting by RNA interference the DPH2 gene transcript is incorporated into: a double stranded RNA, a short hairpin RNA (shRNA) or a microRNA (shRNAmir).
In a further embodiment, the at least one nucleotide sequence targeting by RNA interference the DPH2 gene transcript comprises an antisense strand comprising at least 19 continuous bases of mRNA complementary to the DPH2 gene sequence set forth in SEQ ID No.: 1 and a matching sense strand.
In an embodiment, the at least one nucleotide sequence targeting by RNA interference the DPH2 gene transcript comprises an antisense strand of 19-25 continuous bases, preferably 19-22 continuous bases, of mRNA complementary to the DPH2 gene sequence set forth in SEQ ID No.: 1 and a matching sense strand.
According to a further preferred embodiment of the instant description, the at least one nucleotide sequence targeting by RNA interference the DPH2 gene transcript is selected from i) a double strand RNA composed of an antisense strand comprising a base sequence set forth in SEQ ID No.: 2, and a matching sense strand, the matching sense strand preferably comprising a base sequence set forth in SEQ ID No.: 3, which optionally has an overhang at the terminal of the antisense strand and/or sense strand, and ii) a double strand RNA composed of an antisense strand comprising a base sequence wherein one to several bases have been added to and/or deleted from the 5′ terminal and/or 3′ terminal of the base sequence described in SEQ ID No.: and a matching sense strand, the matching sense strand preferably comprising a base sequence set forth in SEQ ID No.: 3, which optionally has an overhang at the terminal of the antisense strand and/or sense strand.
According to a further embodiment of the instant description, the at least one nucleotide sequence targeting by RNA interference the DPH2 gene transcript is operably linked to at least one regulatory sequence active in a human cell.
In a preferred embodiment, the regulatory sequence comprises a promoter sequence and/or a terminator sequence active in a human cell, being the promoter sequence a constitutive or foreign promoter sequence.
In a different embodiment, the present description concerns an isolated transgenic diphtheria toxin resistant human cell comprising at least one expression vector containing at least one nucleotide sequence targeting by RNA interference the human DPH2 gene transcript, wherein transcription of the at least one expression vector silences the DPH2 gene in the isolated human cell.
According to an embodiment, the at least one expression vector further comprises at least one additional nucleotide sequence.
According to a further embodiment, the instant description concerns use of an expression vector containing at least one nucleotide sequence targeting by RNA interference the DPH2 gene transcript of a human cell for inducing resistance to diphtheria toxin in a human cell, wherein the vector is used in vitro and/or in vivo in a rodent, preferably a mouse, xenografted with a human cell, wherein the transcript of the expression vector is permanently transduced in the human cell, whereby the permanently transduced human cell survives upon diphtheria toxin treatment.
In an embodiment, the expression vector is transduced into a human cell and subsequently the transduced human cell is xenografted into a rodent, preferably a mouse, for in vivo studies of the human cell. Preferably the human cell is a patient derived tumor cell.
In an embodiment, the expression vector contains at least one additional nucleotide sequence, whereby the permanently transduced human cell surviving upon diphtheria toxin treatment contains the at least one additional nucleotide sequence.
According to different embodiments, the at least one additional nucleotide sequence is selected from: i) nucleotide sequences encoding a fluorescent or bioluminescent protein, ii) nucleotide sequences encoding an enzymatic protein marker, iii) nucleotide sequences promoting loss-of-function of an endogenous gene of the permanently transduced human cell, and iv) nucleotide sequences promoting gain-of-function of an endogenous or exogenous gene in the permanently transduced human cell.
In an embodiment, when the at least one additional nucleotide sequence encodes a fluorescent or bioluminescent protein such a nucleotide sequence allows for imaging the permanently transduced human cell and/or monitoring response of the permanently transduced human cell to biological or chemical compounds, or physical treatments.
In a different embodiment, when the at least one additional nucleotide sequence promotes loss-of-function of an endogenous gene of the permanently transduced human cell, or promotes gain-of-function of an endogenous or exogenous gene in the permanently transduced human cell, such a nucleotide sequence allows for defining the biological function of the endogenous and/or exogenous gene in the permanently transduced human cell.
According to a further embodiment, the instant description provides for use of an expression vector containing at least one nucleotide sequence targeting by RNA interference the DPH2 gene transcript of a human cell for inducing resistance to diphtheria toxin in a human cell, wherein the transcript of the expression vector is permanently transduced in the human cell, whereby the permanently transduced human cell survives upon diphtheria toxin treatment for tumor marking, clinical evaluation of anti-tumor therapeutic sequences and/or identification of new oncogenic markers.
In an embodiment, the instant disclosure concerns a method for in vivo selection of human cells (preferably patient derived tumor cells) xenografted in a rodent (preferably a mouse), wherein the human cells are rendered resistant to DT by using an expression vector containing at least one nucleotide sequence targeting by RNA interference the DPH2 gene transcript of the human cells, wherein the transcript of the expression vector is permanently transduced in the human cells, whereby the permanently transduced human cells survive upon diphtheria toxin treatment.
In a preferred embodiment, the method for in vivo selection of human cells xenografted in a rodent provides for using an expression vector further containing at least one additional nucleotide sequence selected from: i) nucleotide sequences encoding a fluorescent or bioluminescent protein, ii) nucleotide sequences encoding an enzymatic protein marker, iii) nucleotide sequences promoting loss-of-function of an endogenous gene of the permanently transduced human cell, and iv) nucleotide sequences promoting gain-of-function of an endogenous or exogenous gene in the permanently transduced human cell.
In a further preferred embodiment, the method for in vivo selection of human cells xenografted in a rodent provides for using an expression vector further containing at least one additional nucleotide sequence selected from: i) nucleotide sequences encoding a fluorescent or bioluminescent protein, and ii) nucleotide sequences encoding an enzymatic protein marker, wherein the at least one additional nucleotide sequence allows for detecting the human cells in the rodent.
In an embodiment, the human cells are transduced with the expression vector either before (i.e. in vitro) or after (i.e. in vivo) being xenografted in the rodent.
Within the present description, the expression “matching sense strand” means reverse complementary sequence, accepting a limited number of mismatches as commonly found in double stranded RNAs, short hairpin RNAs or microRNAs.
The present inventors reasoned that, in principle, injection of a viral expression vector into the patient-derived cancer xenograft (PDX) mass followed by treatment of the mouse with a drug exerting species-specific killing activity only on human non-transduced cells would enable selection of stably transduced PDXs, for marker studies or for proof-of-concept therapeutic target validation.
The present inventors considered that diphtheria toxin (DT) has the required pharmacological properties: it invariably kills human cells using the ubiquitously expressed transmembrane heparin-binding EGF-like growth factor (HBEGF) as a receptor, but has no effect on mouse tissues, because the murine Hbegf does not bind DT. Moreover, DT has been successfully employed for cell lineage ablation in animal models. A well-documented strategy to induce DT resistance in human cells is the blockade of Diphthamide Biosynthesis Protein 2 (DPH2), either by expression of a dominant-negative protein (1) or by gene inactivation via insertional mutagenesis (2). However, both approaches to DPH2 blockade are not optimal resistance markers: (1) expression of a dominant-negative protein would require insertion in the expression vector of a promoter or internal ribosome entry site, typically longer than 500 nucleotides, plus the dominant-negative DPH2 full coding sequence (398 aminoacids, i.e. 1194 nucleotides), accounting in total for more than 1700 nucleotides, which would limit the size of additional elements to be inserted in the vector; (2) insertional mutagenesis is a random, non-directed event, and therefore cannot be programmed to occur in all transduced cells.
DPH2 catalyzes a key step in diphthamide biosynthesis, a histidine modification process known to occur only on His715 of Eukaryotic Elongation Factor 2 (EEF2). After HBEGF-mediated DT internalization, DT inhibits EEF2 by catalyzing the transfer of NAD+ to diphthamide. In the absence of active DPH2, His715 is not converted to diphthamide and the cell is insensitive to DT. Additional genes, including DPH5, DPH6, and DPH7, have been shown to encode proteins essential for diphthamide formation and DT-mediated toxicity in human cells. Notably, cells lacking diphthamide have no distinct phenotypes except their resistance to DT. Therefore, the interruption of diphthamide biosynthesis represents an attractive strategy to render human cells resistant to DT without major biological consequences.
To develop a selectable marker conferring resistance to DT, the present inventors considered an RNA interference approach, using—as an embodiment—a short hairpin sequences inserted into a primary microRNA transcript backbone (shRNAmirs). This design adds a Drosha processing site to the hairpin construct that has been shown to greatly increase knockdown efficiency (3). One or two different shRNAmir sequences were tested for each of the key diphthamide biosynthesis genes.
The present inventors demonstrated that an artificial shRNAmir sequence targeting the DPH2 transcript invariably confers resistance to DT in human cells, and that this sequence can be effectively exploited to select permanently transduced human cells both in vitro and in vivo.
According to a preferred embodiment, the shRNAmir sequence adopted by the present inventors and identified in the following as DTR sequence (SEQ ID No.: 26 also shown in
At odds with the above described features, currently used selectable markers encode for proteins capable of rendering transduced cells resistant to a toxic compound. The coding sequence for such markers is several hundred nucleotides long, and requires a dedicated promoter or an internal ribosome entry site for efficient translation, which substantially increases vector size. This in turn reduces the size of additional genetic elements that can be efficiently cloned and transduced.
A straightforward way to render human cells resistant to DT would be to abrogate expression of HBEGF, encoding the transmembrane DT receptor. However, due to its mitogenic activity and to its release as a soluble form after membrane shedding, its silencing is likely to induce phenotypic changes in both transduced and surrounding cells. Similarly, other enzymes required for diphthamide formation, like DPH1 and DPH3, are not suitable candidates because they have been involved in the regulation of cell growth and development.
Among the remaining candidate targets, DPH2 was not the only one tested in our screening for DT resistance markers: shRNAmirs were tested against each of the DPH5, DPH6 and DPH7 genes. None of them was capable of inducing detectable DT resistance. Indeed, the extent of target mRNA downregulation was quite limited for most of them.
In vivo stable transduction of human cells with vectors containing nucleotide sequence(s) targeting by RNA interference the DPH2 gene transcript, and further containing other nucleotide sequences, like for example fluorescent or bioluminescent proteins, is expected to significantly improve the spectrum and performance of tumor-tracking applications.
This is particularly true for intraperitoneal and other orthotopic models, where caliper measurements are not suitable, and fluorescence or bioluminescence imaging represent a cost effective, simple approach.
The process to induce resistance to diphtheria toxin in human cells disclosed herein enables in fact robust expression of other co-selected coding sequences, as confirmed by the high levels of GFP signal detected by flow cytometry and fluorescence microscopy in selected cells and xenografts, wherein the growth of DTR-GFP transduced tumors was effectively monitored by in vivo imaging, even when the xenografts were intraperitoneal.
Additional possible applications of the process to induce resistance to diphtheria toxin in human cells disclosed herein include transduction of PDXs also with other biologically or therapeutically relevant sequences, like e.g. cDNAs for proteins modulating drug response, or short RNA hairpins interfering with gene expression of undruggable or poorly actionable oncogenic drivers.
In light of the rapidly increasing availability and use of PDX models for preclinical experimentation, the system to induce resistance to diphtheria toxin in human cells presented here offers an unprecedented opportunity for direct genetic manipulation of human tumor xenografts.
Such transduced xenografts, after in vivo selection with DT, may be useful for several applications, depending on the additional genetic elements introduced in the vector containing nucleotide sequence(s) targeting by RNA interference the DPH2 gene transcript in human cells, as here exemplified:
i. if the additional genetic element is a marker (fluorescent, bioluminescent, enzymatic protein or other): follow by vital imaging local and disseminated tumor growth under various conditions, to monitor the activity of pro- or anti-oncogenic molecules and treatments to which the transduced human cells are exposed;
ii. if the additional genetic element promotes the loss-of-function of an endogenous gene of interest in the transduced cells (by RNA interference, recombination, dominant-negative action, or other): follow local and disseminated tumor growth under various conditions, to define the biological function of the targeted gene of interest (e.g. pro- or anti-oncogenic function), and optionally its interactions with pro- or anti-oncogenic molecules and treatments to which the transduced human cells are exposed;
iii. if the additional genetic element promotes the gain-of-function of a gene of interest in the transduced cells (by cDNA overexpression, recombination, mutation or other): follow local and disseminated tumor growth under various conditions, to define the biological function of the targeted gene of interest (e.g. pro- or anti-oncogenic function), and optionally its interactions with pro- or anti-oncogenic molecules and treatments to which the transduced human cells are exposed.
To the above identified aims, development of efficient multi-module expression vectors will be greatly facilitated by the small size and pri-miRNA nature of DTR.
Results
DPH2 Silencing Renders Human Cells Resistant to Diphtheria Toxin In Vitro.
To validate DT as a selective agent, the present inventors verified its activity on a panel of cell lines of different tissue origin, expressing variable levels of the DT receptor, HBEGF (4). DT activity was indeed always high and independent of HBEGF expression levels (
Table 1 provides details of the gene-specific portions of the shRNAmir constructs employed in the screening. For each shRNAmir, columns report the targeted gene, the lentiviral vector system employed and the mature siRNA antisense sequence, which is the active one guiding the RISC complex to specific mRNA degradation. The DTR construct is highlighted in bold.
Transduced cells were then tested for their response to DT (
Human Cells Transduced with DTR Become Resistant to Diphtheria Toxin In Vivo.
To assess if DTR-transduced cells maintain DT resistance also in the context of a living organism, HCT116 cells transduced with DTR or with a control vector were grown as subcutaneous xenografts in nude mice. When xenografts reached ˜50 mm3, mice were treated with DT or with vehicle for three weeks. As shown in
Conversely, DTR transduced cells resisted to both doses of DT and continued growing in the presence of DT and after its withdrawal (
To verify the possibility of using DTR as a marker for in vivo selection of transduced cells, the present inventors designed the experiment illustrated in
Briefly, DTR-GFP-transduced and control HCT116 cells were mixed in a 1:20 ratio, to obtain a heterogeneous population in which the DTR-expressing, GFP+ fraction was around 5%. The mixed cell population was then implanted and grown in nude mice xenografts in the absence or presence of DT for three weeks, followed by two additional weeks without treatment. As shown in
After two weeks from the end of the treatment, flow cytometry on the explanted tumors revealed a striking enrichment of GFP+ cells in the DT-treated arm (
Altogether, these results show that DPH2 silencing by DTR confers resistance to DT also in vivo and can be exploited for in vivo selection of transduced cells.
In Vivo Transduction and Selection for DTR Expression in Xenografts from Human Cell Lines and Tumors.
Generation of genetically modified xenografts is easily accomplishable with most neoplastic cell lines by in vitro transduction and selection, followed by implant in mice.
In the case of PDXs however, such procedure is typically not applicable or poorly efficient.
The present inventors therefore sought to verify if direct intratumoral injection of the DTR vector in xenografts from human cell lines and patient-derived tumors, followed by DT treatment of mice, could lead to the development of xenografts significantly enriched in transduced cells.
To this aim, HCT116 xenografts in nude mice were directly transduced by intratumoral injection of concentrated lentiviral particles of DTR-GFP or control(scramble)-GFP vector.
After one week, cell suspensions were obtained from a first set of transduced tumors for flow cytometry analysis, which detected a fraction of GFP+ cells around 1% for both vector types (average of 5 measurements=0.97%+/−1.70%). In a parallel set of xenografts, one week after transduction, mice were treated with DT for three weeks, followed by two weeks of suspension, after which DT was maintained until tumor explant (
While all tumors displayed marked shrinkage after three weeks of DT treatment, only DTR-transduced tumors resumed growth after the initial shrinkage. The regrown tumors featured a multinodular mass, suggestive of parallel growth of multiple resistant subclones from the areas of vector injection (
Intriguingly, the high fraction of transduced cells and the DT resistance were stably maintained over multiple passages in mice, also in the absence of DT selective pressure (
As a proof of concept of the efficiency of the DTR vector system in an additional preclinical platform, the present inventors reproduced the same experimental procedure employing human CRC PDXs instead of cell line xenografts.
A preliminary test confirmed that also these PDX tumors were sensitive to DT, independently of their genetic makeup (
The DT selection process was then monitored by in vivo tracking of the GFP signal in the transduced tumors. To reduce the background due to the white fur, the skin surrounding the xenograft was shaved. After about five weeks of selection, the GFP signal became detectable in the tumor mass, and kept increasing in the following weeks (
Efficiency of the DTR selection procedure was confirmed in a second PDX model, transduced with single or multiple injections of DTR-GFP lentiviral particles. During DT selection, single or multiple GFP+ areas became detectable, reflecting growth of GFP+ cells in proximity of the injection sites (
Also in this case, the selection process therefore led to the formation of a growing GFP+ tumor.
Materials and Methods
Cell Culture and Reagents
The following cell lines were purchased from American Type Culture Collection (ATCC): HCT-116 (ATCC CCL-247), A549 (ATCC CCL-185), SNB-19 (ATCC CRL2219) and HME1 (ATCC CRL4010). OVCAR-4 cells were purchased from the Cell Line Repository of the National Cancer Institute's Division of Cancer Treatment and Diagnosis (NCI DCTD). Cell lines were maintained in culture following supplier guidelines, and routinely screened for absence of Mycoplasma contamination using the Venor® GeM Classic kit (Minerva biolabs). The identity of each cell line was checked by Cell ID™ System and by Gene Print® 10 System (Promega), through Short Tandem Repeats (STR) at 10 different loci (D55818, D135317, D75820, D165539, D21511, vWA, TH01, TPOX, CSF1PO and amelogenin). Cells were ordinarily supplemented with FBS at different concentrations, 2 mM L-glutamine, antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) and grown in a 37° C. and 5% CO2 air incubator. In particular, HCT-116 and A549 cell line were maintained in RPMI-1640 medium, supplemented with 10% FBS. SNB-19 and OVCAR4 cells were maintained in DMEM, supplemented with 10% FBS. HME1 cells were cultured in DMEM/F-12 supplemented with 20 ng/mL EGF, 10 μg/mL insulin, and 100 μg/mL hydrocortisone and 10% FBS. Proliferation assays were performed seeding 5-15×103 cells in 24-well plates or 5×104 cells in 6 well plate. After 24 hours, medium was replaced adding DT (D0564, Sigma) reconstituted to 0.5 mL with sterile distilled water. After one week of treatments, cells were fixed with a solution of 3% Paraformaldehyde plus 1% glucose and then stained with 0.05% Crystal Violet in Distilled Water.
Gene Sequences
Human DHP2 gene transcript has the nucleotide sequence set forth in SEQ ID NO.: 1, also disclosed in Genbank under accession number NM 001384.4; human DHP5 gene transcript has the nucleotide sequence disclosed in Genbank under accession number NM 001077394.1; human DHP6 gene transcript has the nucleotide sequence disclosed in Genbank under accession number NM 080650.3; human DHP7 gene transcript has the nucleotide sequence disclosed in Genbank under accession number NM 138778.3.
RNA Interference
For stable silencing, pGIPZ shRNAmir vectors targeting DPH2 (V3LHS_382955) and DPH6 (V3LHS_319554 and V3LHS_319557), together with control pGIPZ scrambled-shRNAmir vectors, were purchased from Dharmacon (http://dharmacon.gelifesciences.com). For inducible silencing, pTRIPZ shRNAmir vectors targeting DPH5 (V3THS_318423 and V3THS_318422) and DPH6 (V2THS_212240 and V3THS_301465), plus control pTRIPZ scrambled-shRNAmir vector were purchased from Dharmacon (http://dharmacon.gelifesciences.com).
Table 2 provides detailed information about extent of target transcripts downregulation achieved in cell lines after transduction with lentiviral shRNAmir constructs, analyzed by quantitative real-time PCR. For each cell line, it is reported the target gene, the clone ID of the lentiviral vector employed, the fraction of residual mRNA and the standard deviation values. Data obtained with the DTR construct are highlighted in bold. Indeed, only few shRNAmirs were found to decrease the target transcripts of more than 50%: one for DPH2 and DPH5, one for DPH6 and none for DPH7. The bottom four lines report the silencing activity of DTR in additional cell lines.
The Open Biosystems protocols were followed for bacterial culture growth and for recombination checks. pGIPZ and pTRIPZ plasmids were purified with Miniprep or Maxiprep kits (Qiagen) and DNA concentration was measured by Nanodrop 1000. All lentiviral constructs were transfected into HEK293T cells plated in a 6-well plate at a density of 5×104 cells per well one day prior to transfection, using Lipofectamine 2000 (Invitrogene). Supernatants were harvested after 48 hours, filtered through a 0.45-μm filter, and used to infect cells in the presence of 4 μg/mL of polybrene. Cells were selected in 2 μg/mL puromycin or directly in diphtheria toxin at the indicated doses. For cells transduced with inducible pTRIPZ vector, shRNAmir expression was induced adding doxycycline (1 μg/mL) in to the growth medium and RFP fluorescence was assessed by FACS analysis before performing growth assay and Q-RT-PCR.
For in vivo injection, supernatants of 7 15-cm plates of transfected HEK293 cells were concentrated by ultracentrifugation. Concentration of the viral p24 antigen was determined by HIV-1 p24 core profile ELISA (PerkinElmer Life Sciences).
Q-RT-PCR
Total RNA was extracted using the miRNeasy Mini Kit (Qiagen), according to the manufacturer's protocol.
The quantification and quality analysis of RNA was performed by Thermo Scientific Nanodrop 1000 and Bioanalyzer 2100 (Agilent). DNA was transcribed using iScript RT Super Mix (BioRad) following the manufacturer's instructions.
Q-RT-PCR was performed in triplicate on ABI PRISM 7900HT thermal cycler (Life Technologies) with SYBR green dye. The sequences of the primers (Sigma Aldrich) used for gene expression analyses of DPH2, DPH5, DPH6 and DPH7 genes are reported in Table 3.
Flow Cytometry
GFP expression analysis of in vitro cultured cells was performed by flow cytometry: cells were trypsinized, diluted in a 1% paraformalhdeide-2% FBS solution, stained with DAPI (D9542, Sigma) and analyzed with FACS flow cytometer (CyAn, DAKO).
Explanted tumors were dissected by bistoury and resuspended in collagenase (C5138, Sigma), Trypsin and Medium 199 (Sigma), for 30 minutes at 37° C. After inhibition of tripsine and centrifugation, pellets were washed with HBSS 5% FBS and resuspended in red blood cell lysis buffer (NH4Cl (155 nM), KHCO3 (10 mM), EDTA (0.1 mM)) for 3 minutes. After incubation with DNAse (5 ug/ml) for 5 minutes at 37° C., cells were counted and 10×106 cells were stained with human HLA-ABC antibody (555555, BD Pharmingen) for 45 min in ice. Subsequently, for flow cytometry analyses, cells were filtered with 40 μm filters and stained with DAPI.
Animal Models and Live Imaging
Female, 6-8 week (20-25 grams) non-obese diabetic/severe combined immunodeficient (NOD/SCID) and CD-1 nude mice were purchased from Charles River Laboratories (Calco, Italy) and maintained in hyperventilated cages.
HCT116 xenografts were established by subcutaneous inoculation of 2×106 cells into the right posterior flank of 5-6 week-old mice. Tumor size was evaluated by caliper measurements and the approximate volume of the mass was calculated using the formula (d/2)2×D/2, where d is the minor tumor axis and D is the major tumor axis.
HCT116 secondary grafts, employed for in vivo transduction, were obtained starting from material explanted from HCT116 primary xenograft: tumors were cut into 25 to 30 mm3 pieces and re-implanted in the flank of the mice.
Each intratumoral injection was performed with approximately 1 mg of lentiviral particles diluted in physiological saline solution. Tumor cells were injected subcutaneously into immunodeficient mice as described above (2×106 cells/mouse).
After 2 weeks, mice bearing tumors of approximately 100 mm3 were selected and were randomly divided into 3 groups of 6 mice each.
In vivo transduction experiments were conducted injecting vectors expressing the shRNAmir sequences and GFP reporter directly into the tumor mass. Single or multiple intratumoral injection of 1 mg of lentiviral particles were performed by a 0.5 ml syringe. The same procedure was employed to transduce cell line-derived xenografts and PDXs. In the latter case, tumor samples were procured for PDX engraftment from patients that provided informed consent, and the study was conducted under the approval of the institutional review board.
For selection of cell xenografts, DT was administered three times a week at the indicated dosages and schedules. For PDX selection, DT was administered three times a week (5 μg/kg) until tumor explant.
In vivo quantitative and qualitative analysis of GFP expression was performed by using the IVISR Lumina II imaging and the Living Image software version 3.0 (Caliper Life Science). The GFP filter sets in the system include emission filter (515-575 nm) and excitation filter (445-490 nm). Images of the mice were generated by setting, respectively, 1.5E9 and 1E10 as minimum and maximum values in the color scale. The ROI measurements were individually marked as regions of interest (ROI) in the Living Image software, setting auto ROI detection with 20 as auto detection threshold.
Immunohistochemistry and Confocal Microscopy
To detect GFP expression, explanted tumors were fixed by immersion in formalin for 24 hours at 4° C. Subsequently, the specimens were treated with 30% sucrose solution to protect them from cryopreservation, then embedded in Optimal Cutting Temperature (OCT) compound (Bio-Optica) and stored at −80° C. until sectioning. Subsequently, 12 μm thick sections were cut with a cryomicrotome (Leica CM 3050 S) and collected in Superfrost Plus Slides (Thermo Scientific). Before staining, microscope slides were treated with PBS to remove OCT and stained with DAPI (Sigma). The fluorescent light emitted by Turbo-GFP and DAPI was evaluated on a Leica TCS SP2 AOBS confocal microscope. Images were generated with the LAS AF Leica Application Suite software (Leica) at 20× of magnification. For IHC, formalin-fixed, paraffin-embedded tissues explanted from PDX were partially sectioned (10 μm thick) using a microtome. 4-μm paraffin tissue sections were dried in a 37° C. oven overnight. Slides were deparaffinized in xylene and rehydrated through graded alcohol to water. Endogenous peroxidase was blocked in 3% hydrogen peroxide for 30 minutes. Microwave antigen retrieval was carried out using a microwave oven (750 W for 10 minutes) in 10 mmol/L citrate buffer, pH 6.0. Slides were incubated with monoclonal mouse anti-human Ki67 (clone MIB-1; DAKO) overnight at 4° C. inside a moist chamber. Immunohistochemically stained slides for Ki67, were scanned with a ×400 objective.
Study Approval
All animal procedures and care administered were approved by the Ethical Committee of the University of Turin and the Italian Ministry of Health.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102015000036514 | Jul 2015 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2016/054293 | 7/19/2016 | WO | 00 |
