In vivo siRNA delivery using SAMiRNA Custom Service

SAMiRNA Custom Services, a complete solution for gene function analysis and target discovery and validation through in vivo siRNA studies

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SAMi-RNA

SAMiRNA™ (Self-Assembled-Micelle-inhibitory-RNA) custom synthesis service provides a customized option to provide a target specific delivery of siRNA in interest in vivo, essential for target validation and therapeutics development. We offer a complete solution for RNAi (gene silencing) studies from siRNA, miRNA, in vivo si-/miRNA delivery using SAMiRNA to all the components and services required for quantitative analyses of targeted genes, using a novel SAMiRNA nanoparticle technology.

Since the inception, Bioneer Corporation has been currently developing SAMiRNA™ drugs for the clinical applications through partnership with pharmaceutical companies. To foster open innovation and new partnership opportunities in various therapeutic areas, we are offering valuable opportunities to utilize this second-generation siRNA technology at an affordable price for translational in vivo siRNA studies. This also includes research and technical support teams ensure the top-quality products and services to meet the unique needs of clients from companies and research institutions worldwide. Our world-class teams have highly profiled expertise in nucleic acid manufacturing and its in vivo applications

For that this service provides a seamless approach to place an order for custom SAMiRNA™ of interest, based on a target gene of interest. Upon manufacturing, the final product is then delivered, which can simply be re-suspended and used for the efficacy testing in animals. Upon customer’s interest, we also provide additional screening in vitro and validation services that include specific siRNA design, in vitro validation of siRNA for further optimization studies and assess knockdown efficiency in vivo. All of our SAMiRNA™ service and products are provided for Research Use Only. For further development, please contact us for the licensing program.


SAMiRNA™ (Self-Assembled-Micelle-inhibitory-RNA)

 One major hurdles in RNA interference (RNAi)-based therapeutics is the proper delivery of siRNA in interest to target tissue and its adverse side effects caused by the specific type of a siRNA delivery vehicle (Table 1). To mediate this, Bioneer has developed SAMiRNA (Self-Assembled-Micelle-inhibitory-RNA), which is a novel single-molecular synthetic siRNA, conjugated with lipid and hydrophilic polymer, spontaneously assembled as a stable nanoparticles (NP) with protective PEG coat and lipid core in the nano-scale size level. This system is optimized for systemic delivery and localization in either vascularized tumors through Enhanced Permeability and Retention (EPR) effect with no other reagents need and formulation process. Such novel approach of synthesis provides solutions for the development of RNAi-based drugs. As a second-generation RNAi drug technology, SAMiRNA is synthesized as a single chemical entity as a form of nanoparticle (NP), manufactured by one-step automated solid phase synthesis requiring no formulation process and no innate immune response.


Table 1: Delivery challenges overcome by SAMiRNA™ technology

Challenges in siRNA delivery SAMiRNA™ system
Rapid clearance and degradation in serum Improved serum stability
Toxicity of delivery systems No detectable liver toxicity or innate immune response
Limited tissue specificity Tumor tissue targeting capabilities
Low silencing potency Long lasting in vivo silencing efficiency


 The therapeutic potential of SAMiRNA™ is highlighted with negligible toxicity, outstanding for its in vivo serum stability, as well as its target gene silencing efficacy in various animal disease models including cancer and lung fibrosis models. In addition, SAMiRNA™ has been shown to induce no innate immune response as demonstrated for a variety of diseases in human PBMC test and high dose-administrated rodent models. This is due to Bioneer’s extensive experience in both nucleic acid chemistry and large scale nucleic acid manufacturing capacity and further advancing into clinical development pipelines with top-quality DNA/RNA oligos and siRNA worldwide for 20 years.


Our SAMiRNA Custom Services include:

  • Custom siRNA synthesis

    • Custom siRNA oligos are offered based on the sequences of your interest. Up to 30-mer siRNA including a choice of 32 different 3' overhangs can be synthesized with a variety of modification options for expanded specificity. With this, SAMiRNA consists of proprietary polymer -conjugated siRNA that is provided purified, annealed, and lyophilized which can simply be resuspended for efficacy tests in animals.

  • Genome-wide pre-designed siRNA

    • Genome-wide predesigned siRNA library is available for mouse, rat or human. Bioneer’s proprietary Turbo-si designer algorithm designs siRNA from your gene of interest that provides superb knock-down efficiencies of specific targets by binding to the structural hindrance-free region and avoiding off target effect.

  • Custom SAMiRNA™ nanoparticle synthesis for in vivo siRNA delivery, and efficacy/biodistribution tests using animal models

    • siRNA contains high lipo-phillic cell membrane barrier and is known to get rapidly degraded when delivered into the blood stream in vivo, triggering undesirable immune responses. Thus, SAMiRNA™ resolves all of the limitations, from studies conducted in solid tumor model.

      The siRNA oligos can be manufactured in the form of SAMiRNA with SAMiRNA™ nanoparticles (NP) can be provided upon request. (see what SAMiRNA™ is here http://www.bioneer.co.kr/SAMiRNA/Platformtechnology.aspx). Upon manufacturing, the reagent can then be re-suspended in PBS and injected these into animals.

  • In vitro screening and validation of siRNAs for lead optimization

    • To achieve and validate siRNA with highest knockdown efficiency, Bioneer recommends “in vitro screening and validation service”, instead of choosing from in silico. This approach is based on constructing and transfecting up to 100 siRNAs into cells individually, aiming specific mRNAs of interest. Its results in the knockdown efficiencies are ranked and analyzed by RT-qPCR. Upon selection, we determine candidates of most potent siRNAs and SAMiRNA nanoparticles (NP) by revalidating in vitro before shipment.

  • Quantitation of functional knockdown of target gene by real time RT-qPCR (Accupower® qPCR Array Service)

    • Bioneer also offers qPCR Array service for quantitation of functional knockdown of target gene by real-time RT-qPCR. The process follows the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. Experts conduct the experiments and provide data in a short period of time.

    • For more information, please visit “qPCR Array Service”. Bioneer’s SAMiRNA™ Services provides a rapid on-line quotation at siRNA@bioneer.com for siRNA; qPCRarray@bioneer.com for Accupower® qPCR array service. Please let us know the details of your project, so that we can provide you with a quotation and a timeline estimate.

  • To order and use custom SAMiRNA™ incorporating customized siRNA or miRNA

SAMiRNA™ (Self-Assembled-Micelle-inhibitory-RNA)

One of the major obstacles in the development of RNA interference (RNAi)-based therapeutics is siRNA/miRNA delivery. SAMiRNA (Self-Assembled-Micelle-inhibitory-RNA) is a novel class of RNAi molecule developed by Bioneer, which provides the solution for virtually all major difficulties in the development of RNAi-based therapeutics including siRNA delivery.

SAMiRNA is prodrug that is manufactured as a single chemical entity and spontaneously Self-Assembles into nanoparticles with a protective PEG coat. SAMiRNA nanoparticles can incorporate siRNA, miRNA or even antisense DNA, and are the ideal size for selective localization in either vascularized tumors, (via Enhanced Permeability and Retention (EPR) effects), or to targeted tissues through the addition of targeting moieties to the surface of SAMiRNA.

The therapeutic potential of SAMiRNA is outstanding because of its negligible toxicity and outstanding in vivo serum stability as well as its efficacy in target gene silencing. Our data demonstrate SAMiRNA's exceptional therapeutic potential as an RNAi platform for a variety of diseases. Bioneer is uniquely positioned for the development and advancement of our SAMiRNA system as it combines our extensive pre-clinical research expertise with our nucleic acid manufacturing and development expertise: Bioneer has been providing top-quality DNA oligos and siRNAs worldwide for 20 years.

Using this novel RNAi prodrug technology, Bioneer is currently advancing clinical development of pipeline programs for previously non-druggable diseases through our own pipeline efforts as well as through partnerships with major global pharmaceutical companies.


Key features of SAMiRNA™

: Best-in-class RNAi drug SAMiRNA overcomes the Unmet needs in siRNA drug development

Unmet needs in siRNA drug development SAMiRNA™
Adverse effects No detectable liver toxicity or innate immune response
Delivery (Issues with other technologies: rapid clearance and degradation in blood, No target tissue specificity, low in vivo efficacy) Outstanding serum stability (PK/PD validated),
tumor/tissue targeting capabilities verified, long lasting in vivo efficacy validated in animal disease models
Manufacturing cost One-step automated solid-phase synthesis of SAMiRNA and rapid chromatographic purification process: enormous advantage allowing for economical large scale production of SAMiRNA.
QC processes Since SAMiRNA is a Single Chemical Entity the QC process is dramatically simplified
Expansion of indications Powerful siRNA delivery platform technology with flexibility to incorporate siRNA (or miRNA or antisense) sequences against any disease target; Your target gene(s) can be easily transformed into SAMiRNA nanoparticle!


SAMiRNA™ Summary

Best-in-class RNAi prodrug technology: SAMiRNA™ is the most unique and effective RNAi prodrug developed to date for the treatment of cancer and other diseases.


Pre-clinical data for SAMiRNA

Novel self-Assembling siRNA nanoparticles with a protective PEG coat and optimal monodisperse nano-
   scale size(Figure 1)
siRNA Prodrug: highly stable in circulation with siRNA release and activity only after metabolism within
   target cells
Outstanding serum stability (Figure 4)
in vivo efficacy validated in animal disease models: completion of preclinical tests for cancer treatment
   (Figure 2)
Extremely low toxicity and cytokine induction (Figure 5-7)
Combined with various targeting moieties, SAMiRNA nanoparticles can target various organs of interest,
   for example, liver

A new class of single molecule-based self-assembling Nanoparticles

Figure 1. Structure of SAMiRNA Nanoparticle.
(A) Schematic diagram of SAMiRNA (B) Cryo-TEM images of SAM-siRNA Nanoparticles (scale bar = 100 nm).

Long-lasting in vivo efficacy validated in mouse cancer models

Figure 2. in vivo silencing of target mRNA by SAMiRNA Nanoparticels.
(A) Tumor bearing mice were intravenously injected with saline (PBS), or with a single 5mg/kg dose of either survivin-SAMiRNA, or tumor-targeting folic acid (FA)-conjugated survivin-SAMiRNA. Mice were sacrificed at denoted time points and survivin mRNA levels in isolated tumor cell masses were subsequently measured using Real-Time PCR. (B) Tumor bearing mice were intravenously injected with saline (PBS) and survivin-SAMiRNA NP at a single 1 or 5mg/kg dose. Mice were sacrificed at 72 hr postinjection and survivin mRNA levels in isolated tumor cell mass were subsequently measured using real-Time PCR.

Tumor-specific targeting of SAMiRNA in mouse cancer models

Figure 3. in vivo targeting of SAMiRNA Nanoparticles to s.c. grafted tumor.
(A) Time-dependent in vivo tumor targeting specificity of Cy5.5-labeled SAMiRNA, delivered via i.v. to tumor-bearing nude mice. (B) Images of various organs extracted from treated mice 12 hours after injection. No fluorescence was detectable outside of the tumor proper.

in vivo PK/PD Quantification of i.v. administrated SAMiRNA™

Shows significantly enhanced stability compared to modified siRNA (2'-O-Methyl siRNA)



Figure 4. Quantification of siRNA in plasma from mice i.v. injected with SAMiRNA.
Mouse blood was extracted at indicated time points (hours post-injection) and SAMiRNA and siRNA levels in isolated plasma were measured by qRT-PCR assay. The linear regression of the amplification curve shows an excellent R2 value for this assay.

SAMiRNA™ has no detectable adverse effects

Did not trigger any IFN response or liver toxicity

Figure 5. General toxicity test of SAMiRNA
Naked siRNA and SAMiRNA were i.v. injected into ICR mice in order to evaluate toxicity. Serum was collected 6 or 24 hr post-injection and toxicity markers were analyzed. None (not-injected), ICR 5 weeks (average value from normal 5-week old ICR mice, from published literature), b-gal 728 (beta-galactosidase siRNA), b-gal 728 with TF (beta-galactosidase siRNA mixed with in vivo MegaFectine from QBiogene), NC siRNA (negative control siRNA), Sur584 (survivin siRNA), SAM-sur584 (survivin siRNA-containing SAMiRNA).

SAMiRNA™ has no detectable adverse effects

Negligible cytokine induction in human PBMCs by SAMiRNA

Figure 6. Cytokine response to SAMiRNA in human PBMC
Human whole blood was incubated with 0.1 or 1 uM of naked siRNA or SAMiRNA for 24 hrs at 37°C and the resulting supernatant was measured for various cytokine induction. Concanavalin A (ConA) was used as positive control.

SAMiRNA™ has no detectable adverse effects

Extremely low toxicity profile of SAMiRNA
Independent results from a GLP-certified CRO, Biotoxtech Co. Ltd)




Figure 7. in vivo SAMiRNA blood chemical and hematological analysis
10 mpk of SAMiRNA, naked or modified siRNA was repeatedly i.v. injected to ICR mice (n=5) 3 times at intervals of one day.
Whole blood and separated serum were collected and analyzed 24 hrs after the final injection.

Toxicity test of SAMiRNA-Survivin including 28-day repeated dose

1. General Toxicology Study

  1) Acute toxicity (100mpk, single dose)
      - Body weight changes
      - Pathology findings
  2) Repeated dose 28-Day (25, 50, 100mpk, multiple dose)
      - Body weight changes
      - Hematology parameters / Clinical chemistry parameters
      - Urinalysis parameters
      - Necropsy findings / Organ weighed / Tissue preserved
      - Histopathological findings

2. Genetic Toxicology Study

  1) Ames Test (5,000 ug per plate)
      - Signs of toxicity
      - Individual plate counts
      - Dose-response relationship
      - Statistical analyses
      - Concurrent negative (solvent/vehicle) and positive control data, with ranges, means and standard
         deviations
      - Historical negative (solvent/vehicle) and positive control data, with e.g. ranges, means and standard
         deviations
  2) Chromosome Aberration Assay (5,000 ug per plate)
      - Cytotoxicity measurements
      - Signs of precipitation
      - Data on pH and osmolality of the treatment medium
      - Definition for aberrations, including gaps
      - Changes in ploidy
      - Concentration-response relationship
      - Statistical analyses
      - Concurrent negative (solvent/vehicle) and positive control data
      - Historical negative (solvent/vehicle) and positive control data, with ranges, means and standard
         deviations

Safety of SAMiRNA: 4-week repeated dose tox profiling
Results

1. General Toxicology Study

  1) Acute toxicity
      - No acute toxicity including bodyweight changes and histological abnormalities were observed
  2) Repeated dose 28-Day
      - No clinically significant or dose-dependent changes were observed post-treatment in
        hematology, chemistry, urinalysis, coagulation parameters, reticulocyte counts, complement levels
      - NOAEL (No Observed Adverse Effect Level) of SAMiRNATM was 100mg/kg/day

2. Genetic Toxicology Study

  1) Ames test
      - No significant induction for mutagenic activity was observed with highest dose tested,
         5,000ug per plate
  2) Chromosome aberration assay
      - No significant aberrations in chromosomes were noticed with highest dose tested, 5,000ug per
         plate

Safety Assessment

Safety evaluation through 28-day repeated dose and genetic tox studies demonstrated that SAMiRNA is
an exceptionally safe and efficient drug candidate

Safety of SAMiRNA: No adverse effects

Extremely low toxicity profile of SAMiRNA
(by a GLP-certified CRO, Korea Institute of Toxicology)





Manual


MSDS

MSDS_In vivo siRNA delivery using SAMiRNA Custom Service


Brochure

• SAMiRNA Custom Service


Quality Assurance

Bioneer is the holder of Quality Management System Certificates for the following standards.

• ISO 9001 – certificate



Custom Designed SAMiRNA Order From

Bioneer’s SAMiRNA Custom Services will be provided through a SAMiRNA ordering system with comprehensive service packages at highly competitive prices. Please let us know the details of your project, so that we can provide you with an accurate quotation and timeline estimate


SAMiRNA ordering

SAMiRNAs are synthesized by incorporating siRNA sequences into SAMiRNA platform. siRNA sequences can be picked from either custom siRNAs or Bioneer’s pre-designed siRNA pool .

1. Custom siRNA (link to ordering form)
SAMiRNAs loaded with customized siRNAs are available. Please let us know a sequence of your own functional anti-sense strand. SAMiRNA nano-particle of duplex annealed with a corresponding complementary sequence will be provided.

2. Pre-designed siRNA (link to ordering form)
Pre-designed siRNA sequences for SAMiRNA synthesis can be searched in the Bioneer AccuTarget Pre-designed siRNA database by Gene ID, Symbol, Synonyms, Description and/or Accession numbers.

3. SAMiRNA controls (link to ordering form)
Varieties of SAMiRNA controls are available to confirm the functional activities and/or the delivery of SAMiRNA to target tissues. SAMiRNAs constituted with siRNA sequences aiming at non-target (negative control), GFP and luciferase are provided. In addition, SAMiRNAs can be labeled with various fluorescent dyes to detect the localization of SAMiRNA in vivo animal models.

  • The formation of SAMiRNA nano-particle will be guaranteed upon re-suspension by providing QC data regarding the size and PDI information of the nano-particle.

SAMiRNA Order scale of SAMiRNA (nmole) Number of mouse
(intravenous injections)
Price
Custom Designed SAMiRNA   100   5mg/kg -> 5 mouse
  1mg/kg -> 25 mouse
  Inquiring Only
  500   5mg/kg -> 25 mouse
  1mg/kg -> 125 mouse
  1000   5mg/kg -> 50 mouse
  1mg/kg -> 250 mouse
Control SAMiRNA   100   5mg/kg -> 5 mouse
  1mg/kg -> 25 mouse
  Inquiring Only
  500   5mg/kg -> 25 mouse
  1mg/kg -> 125 mouse
  1000   5mg/kg -> 50 mouse
  1mg/kg -> 250 mouse
Fluorescence conjugated SAMiRNA

(FITC, Cy5.5 etc)
  100   5mg/kg -> 5 mouse
  1mg/kg -> 25 mouse
  Inquiring Only
  500   5mg/kg -> 25 mouse
  1mg/kg -> 125 mouse
  1000   5mg/kg -> 50 mouse
  1mg/kg -> 250 mouse
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