RNAi Nanoparticle System for Murine Cancer Models
Use InVivoPlex® to:
- Study in vivo function of tumor genes in a clinically relevant fashion
- Prioritize the most promising gene targets for drug discovery
- Study multi-gene inhibition or gene interactions with chemotherapy
- Validate candidate RNAi or gene cancer therapeutics in vivo
InVivoPlex® self-assembling, RNAi nanoparticles form with a protective coat and optimal size for selective localization in vascularized tumors through an Enhanced Permeability and Retention mechanism (EPR). The easy to use kit provides everything needed to use the polymeric reagent to form the nanoparticles with your in vitro optimized RNAi, siRNA, shRNA, plasmids or even combinations. The reagent can reveal in vivo activity of candidate therapeutic targets, simulating effects of an antagonist drug before costly drug discovery.
- Simple to use kit that forms siRNA nanoparticles <50 nm, and stable for 1 week
- Highly selective uptake by vascularized murine tumor models, minimual lung uptake
- Low toxicity and low levels of cytokine induction, minimizing off-target effects
- Clinical relevance: multiple dose regimens, dose response, effect simulates targeted therapy
- Delivers siRNA oligonucleotides, plasmids, and combinations
Tumor Targeted, Therapeutic RNAi Nanoparticle Capabilities
- Tumor Selectivity: Delivery is directed to vascularized tumor tissue, away from organs
- Repeat Dosing: Administer 3 times per week, without toxicity
- Dose Response: Evaluate dosages from 1 mg/kg to 6 mg/kg
- Many Models: Many murine cancer models exhibit EPR active tumors
- Multiple Cargos: siRNA, plasmids, and combinations can be delivered
Top: InVivoPlex® luciferase plasmid nanoparticles given iv selectively target vascularized subcutaneous tumors in xenograft murine cancer models, without complicating lung or other organ activity typical of non-targeted in vivo transfection reagents.
Bottom: InVivoPlex® siRNA nanoparticle treatment by iv, 3 times per week for two weeks, inhibits xenograft tumor growth. Left: pan human siRNA (Qiagen). Right: InVivoActTM hVEGF – equivalent to inhibition by the approved hVEGF mAb therapeutic.
Simple to Use , Complete Kit
- Simple Assembly: Add aqueous nucleic acid to NP Reagent Vial drop wise while stirring
- Cargo Flexibility: You select the cargo: siRNA, shRNA, or expression plasmid
- Convenient: Nanoparticles can be prepared once each week for administration
- Complete Kit: Mixing system and all buffers, including Vehicle Control, are provided
- Accessory Kits: Specialized RNAi to facilitate tumor uptake and gene activity studies
Points To Consider
Determine murine tumor model EPR activity:
Studies are recommended to first evaluate EPR activity of tumor models, or to adjust tumor model parameters to increase EPR activity. This can be accomplished using InVivoTrak™, fluorescent dye labeled agents designed for tumor uptake studies, or InVivoPlex® with a reporter gene plasmid. Initial studies with these agents can address concerns of variability of cancer models among labs, and variation related to tumor vasculature that influence activity of the nanoparticle targeting the tumor tissue. The parameters of the protocol can be established for efficacy studies, confirming that InVivoPlex® nanoparticles actually target oligonucleotide delivery selectively to the tumor tissue.
Use InVivoPlex® to evaluate gene or protein inhibition in vivo:
Use of InVivoPlex® to evaluate gene and protein inhibition will likely require repeated treatments, and is expected to give 20-40% inhibition of targeted mRNA. We recommend three doses, e.g. Monday, Wednesday, and Friday, assayed the following Monday. Note, even modest inhibition of key proteins can elicit a significant effect – see the figure on page 2, showing equivalent inhibition of an NSCLC xenograft tumor model by Avastin® or InVivoPlex® delivery of InVivoActTM–hVEGF. The genes and proteins showing the greatest effect from low levels of inhibition are likely to represent the most potent points for therapeutic intervention.
Evaluate experimental therapeutic targets in vivo with InVivoPlex®:
Use of InVivoPlex® to evaluate candidate therapeutic targets identified in vitro with RNAi agents will usually require repeated doses to show an effect on tumor behavior, and typically requires multiple treatment groups with negative controls and ideally a positive control RNAi agent. InVivoActTM control RNAi have been developed to provide negative and positive controls, as shown in results on page 2 where InVivoPlex® with InVivoActTM–hVEGF inhibited H460 tumor growth as well as the approved mAb therapeutic while InVivoActTM–Neg had no effect compared to buffer control treatment. These agents can be used to facilitate validation of candidate targets with even greater potency in similar tumor cell pathways.
Use InVivoPlex® to evaluate gene combinations or interaction with chemotherapeutics:
The ratio of siRNA or plasmid combinations for multi-target studies will depend on their relative potency, e.g. plasmid expression or siRNA gene inhibition activity. Combination studies can be initiated with equal amounts in studies characterizing activity, permitting adjustment of the ratio based on relative potency. When combining plasmid gene expression with siRNA gene inhibition, we found that about one third siRNA by weight provided both gene expression and plasmid gene inhibition by an inhibitory siRNA, after 24 hours. To study synergy or interference of target genes with chemotherapy, multidose regimen studies can be performed by including groups with separate and combined siRNA and chemotherapy treatments.
Regulatory issues for performing InVivoPlex® studies:
Use of animals for scientific research usually is subject to national and in some cases regional or local laws. For laboratories in the US AparnaBio can help provide draft protocol language and guidance as well as links to information available from the US Government, and gladly discuss how use of InVivoPlex® can be described to review committees, including a description of how its use is consistent with established humane use of experimental animals.
InVivoPlex® Kits enable translation of RNAi studies to murine models of cancer, with the objective to allow studies producing clinically relevant information on tumor gene function, candidate targets for therapeutic intervention, and in vivo validation of RNAi therapeutic candidates. The InVivoPlex® Kits supply all the required buffers and the nanoparticle forming reagent, along with everything needed for specialized mixing with user supplied RNAi agents forming EPR targeted nanoparticles that localize in vascularized tumors in murine models of cancer, as well as vehicle control buffer for a control treatment group of mice. An important advantage of the reagent is the feature that it allows the user to prepare the nanoparticles with their experimental RNAi agent, and the capability to use a wide variety of RNAi agents as well as to perform combination RNAi studies. InVivoPlex® Kits are available for preparation of nanoparticle samples for treatment of small, medium, or large groups of mice. Kits for other group sizes can be provided upon request.
To use InVivoPlex®, we recommend starting with a tumor uptake study to insure EPR activity of the specific murine cancer model to be used, or its optimization for EPR activity. The InVivoTrakTM fluorescent labeled companion products are available to facilitate these studies. The InVivoTrakTM is available with either red or near IR fluorescent labels, and can be used with tumor homogenates in a fluorescence plate reader or with an NIR in vivo imaging instrument. InVivoTrakTM fluorescent tracer products are designed for use with small size group studies. The InVivoTrakTM Oligo products are designed for use with the small InVivoPlex® kit, to make fluorescent labeled nanoparticles for testing EPR tumor uptake. InVivoTrakTM is available with either red or near IR fluorescent labels. The InVivoTrakTM Carrier products are designed for use with unlabeled RNAi or with InVivoTrakTM oligo products for dual label tracer studies.
If studies of tumor gene inhibition with InVivoPlex® are desired, we recommend a one week, three dose protocol.
For studies of RNAi activity on tumor growth or other phenotype, we recommend multi-dose studies over several weeks with multiple treatment groups that include negative and positive controls. The InVivoActTM negative and positive RNAi agent companion products are available to facilitate these studies. InVivoActTM products are designed for use with the large InVivoPlex® Kit, and are available in forms to provide a negative RNAi treatment and to provide positive RNAi treatments that inhibit a clinically validated therapeutic target gene, such as VEGF or EGFR.
For more information, please contact us by phone or email.