sdASO™ In Vivo Protocol
Comprehensive protocol for self-delivering antisense oligonucleotides in animal models
Introduction
This protocol provides detailed instructions for using AUM Biotech's self-delivering antisense oligonucleotides (sdASO™) in animal models. Our sdASO™ products enable efficient RNA target modulation in vivo without specialized delivery vehicles, making them ideal for a wide range of research applications.
Protocol Overview
AUM Biotech's sdASO™ in vivo application is a straightforward process:
- Prepare sdASO™ formulation in appropriate buffer
- Select appropriate administration route
- Administer to study animals at calculated dosage
- Monitor and collect samples at optimal timepoints
- Analyze target knockdown in tissues of interest
This protocol can be adapted for different animal models, administration routes, and study endpoints based on your specific research needs.
- No Specialized Delivery Required: Unlike traditional ASOs or siRNAs, our sdASO™ products do not require complex formulations or conjugation for in vivo efficacy
- Multiple Administration Routes: Effective via intravenous, subcutaneous, intraperitoneal, intrathecal, and other routes
- Broad Tissue Distribution: Accumulates in multiple organs and tissues
- Minimal Immunogenicity: Low risk of triggering innate immune responses compared to unmodified oligonucleotides
Materials and Reagents
Required Items
- AUM Biotech sdASO™ (lyophilized or stock solution)
- Sterile formulation buffer (PBS, saline, or specialized buffer)
- Administration equipment:
- Sterile syringes and needles (appropriate gauge for selected route)
- Alcohol swabs
- Restrainers (if needed for proper animal handling)
- Animal subjects (appropriate for study design)
- Sample collection materials:
- Surgical tools (for tissue collection at endpoint)
- Blood collection tubes/needles (if performing PK or monitoring)
- RNase-free collection tubes
- RNA preservation reagent (e.g., RNAlater™ or equivalent)
- Liquid nitrogen (for snap freezing samples)
- Analysis materials:
- RNA extraction kit
- qRT-PCR reagents or other RNA analysis tools
- Protein extraction buffers (if analyzing protein knockdown)
- Western blot or ELISA reagents (if applicable)
Detailed Protocol
Animal Preparation
Prepare animals according to institutional guidelines and study requirements.
Ensure animals have acclimated to facility conditions for at least 5-7 days prior to study initiation.
Record baseline measurements (weight, behavior, clinical parameters) before administering sdASO™.
Group animals appropriately based on study design (treatment groups, controls, etc.).
Fast animals if required by your administration protocol (typically not necessary for most routes).
All animal studies should be conducted in accordance with institutional animal care guidelines and with proper ethical approvals. Ensure that personnel are properly trained in animal handling and administration techniques.
sdASO™ Formulation Preparation
Prepare sdASO™ formulation at the appropriate concentration for your selected administration route and dosage.
Resuspend lyophilized sdASO™ in sterile PBS, saline, or appropriate buffer to the desired concentration.
For most applications, concentrations of 1-10 mg/mL are suitable for in vivo administration.
Calculate the required volume based on animal weight and target dose (typically 3-30 mg/kg for mice, see dosing table below).
Filter sterilize the solution through a 0.22 μm filter if possible.
Prepare formulations fresh on the day of administration when possible, or store at 4°C for no more than 24 hours.
Avoid multiple freeze-thaw cycles of sdASO™ formulations. For studies requiring multiple dosing days, prepare fresh formulations each day or store aliquots at -20°C and thaw only once before use.
sdASO™ Administration
Administer the sdASO™ formulation using the appropriate route for your target tissue and research objectives.
Intravenous (IV) administration: For broader systemic distribution, inject into the tail vein (mice) or appropriate vein according to species. Use a maximum volume of 5-8 mL/kg and inject slowly (over 20-30 seconds).
Subcutaneous (SC) administration: Inject into the loose skin of the neck or flank. Maximum recommended volume is 10 mL/kg for mice.
Intraperitoneal (IP) administration: Inject into the peritoneal cavity after proper restraint. Maximum recommended volume is 10 mL/kg for mice.
Intrathecal (IT) administration: For CNS targeting, inject directly into the intrathecal space. This requires specialized training and typically uses volumes of 5-10 μL in mice.
Other routes: sdASO™ can also be administered via local injection to specific tissues (e.g., intramuscular, intraocular, intracerebral) depending on research needs.
Route selection should be based on your target tissue. IV administration typically yields good distribution to highly perfused tissues (liver, kidney, heart), while SC provides more sustained release. For CNS applications, IT administration provides the best access across the blood-brain barrier.
Monitoring and Sample Collection
Monitor animals post-administration and collect samples at appropriate timepoints.
Monitor animals for any adverse reactions immediately after administration and at regular intervals throughout the study.
Collect blood samples at predetermined timepoints if pharmacokinetic analysis is part of your study.
For terminal timepoints, collect target tissues according to your study design. Typical timepoints for assessing maximal knockdown are 48-96 hours post-administration for mRNA targets, and 72-120 hours for protein targets.
Process each tissue appropriately:
- For RNA analysis: Either snap freeze in liquid nitrogen or preserve in RNA stabilization reagent
- For protein analysis: Snap freeze or prepare fresh lysates as appropriate
- For histological analysis: Fix in appropriate fixative (e.g., 4% paraformaldehyde)
The kinetics of knockdown vary by tissue and target. For liver targets, mRNA knockdown is typically observed within 24-48 hours, while CNS targets may take longer (72-96 hours). Multiple timepoints are recommended for initial studies to establish optimal analysis timepoints.
Target Knockdown Analysis
Analyze collected samples to evaluate the efficacy of sdASO™-mediated target knockdown.
Extract RNA from tissue samples using an appropriate RNA isolation method. Maintain RNase-free conditions to ensure sample quality.
Perform qRT-PCR to quantify target RNA levels relative to untreated controls and normalized to appropriate housekeeping genes.
For protein targets, extract proteins and perform Western blot, ELISA, or other appropriate protein quantification methods.
Analyze functional endpoints relevant to your study (e.g., behavioral assessments, physiological measurements, biochemical parameters).
If using fluorescently labeled sdASO™, tissue distribution can be visualized using fluorescence microscopy on frozen sections.
It is strongly recommended to include both dose escalation and time course components in pilot studies to determine the optimal dose and timepoint for your specific target and tissue. Knockdown efficacy can range from 40-95% depending on the target, tissue, dose, and administration route.
Reference Guidelines
The tables below provide guidelines for dosing and administration routes:
| Animal Model | Typical Dose Range | Starting Dose | Maximum Recommended Dose |
|---|---|---|---|
| Mouse | 3-30 mg/kg | 5 mg/kg | 50 mg/kg |
| Rat | 2-25 mg/kg | 3 mg/kg | 40 mg/kg |
| Zebrafish (embryo) | 0.1-0.5 nmol/injection | 0.2 nmol/injection | 1 nmol/injection |
| Non-human primate | 1-10 mg/kg | 2 mg/kg | 20 mg/kg |
| Administration Route | Target Tissues | Advantages | Considerations |
|---|---|---|---|
| Intravenous (IV) | Liver, kidney, spleen, lung, heart | Rapid distribution, high liver uptake | Requires skilled technique, limited CNS distribution |
| Subcutaneous (SC) | Systemic with prolonged exposure | Easy administration, sustained release | Slower onset than IV, may require higher doses |
| Intraperitoneal (IP) | Liver, kidney, intestine, peritoneal tissues | Relatively easy administration | Variable absorption, potential for incorrect administration |
| Intrathecal (IT) | CNS (brain, spinal cord) | Direct CNS access, bypasses BBB | Technically challenging, specialized training required |
| Intramuscular (IM) | Muscle tissue | Local targeting of muscle tissue | Limited distribution beyond injection site |
- The doses provided are general guidelines; optimal doses may vary depending on the specific sdASO™, target, and application.
- For initial studies, a dose-response approach is recommended (e.g., 3, 10, and 30 mg/kg in mice) to determine the optimal dose for your target.
- For long-term studies, repeated dosing may be necessary. Typical maintenance regimens include weekly or biweekly administrations at the established effective dose.
Tips and Troubleshooting
Optimization Tips and Best Practices
Pilot Studies
Begin with small pilot studies incorporating both dose escalation and time course components. This will help establish optimal dosing, administration routes, and sampling timepoints before proceeding to larger studies.
Multiple Controls
Include proper controls: untreated animals, vehicle-only, non-targeting/scrambled sdASO™, and positive controls (if available). This comprehensive control strategy helps differentiate specific knockdown effects from non-specific responses.
Tissue Collection
For optimal RNA quality, process tissues immediately after collection. Either snap-freeze in liquid nitrogen or place in RNA stabilization solution within minutes of collection. Avoid repeated freeze-thaw cycles of tissue samples.
Multi-level Analysis
Analyze knockdown at multiple levels: mRNA (qRT-PCR), protein (Western blot, IHC, or ELISA), and functional outcomes. This comprehensive assessment provides a complete picture of target modulation efficacy.
Troubleshooting Common Issues
Low or Variable Knockdown Efficiency
- Increase dose: If knockdown is lower than expected, consider increasing the dose. A dose-response study (e.g., 3, 10, and 30 mg/kg) can help determine the optimal dose.
- Optimize administration route: Different routes provide varying distribution patterns. Consider switching routes if target tissues show inadequate exposure.
- Extend time course: Peak knockdown may occur later than anticipated. Collect samples at multiple timepoints (e.g., 24, 48, 72, and 96 hours) to identify peak knockdown.
- Check tissue collection and processing: Ensure tissues are collected and processed properly to maintain RNA quality. Degraded RNA can lead to inaccurate measurements.
- Consider multiple doses: For some targets or tissues, multiple administrations (e.g., daily for 3-5 days or weekly for several weeks) may provide better knockdown than a single dose.
Adverse Reactions or Toxicity
- Reduce dose: If toxicity is observed, reduce the dose or consider alternative administration routes.
- Slow injection rate: For IV administration, injecting too rapidly can cause acute reactions. Ensure injections are performed slowly and steadily.
- Check formulation: Ensure the buffer is appropriate (pH 7.2-7.4) and free of endotoxins or other contaminants.
- Monitor liver and kidney function: ASOs can sometimes affect liver enzymes or kidney function. Consider monitoring these parameters, especially at higher doses.
- Distinguish target-mediated effects: Some adverse effects may be due to successful target knockdown rather than toxicity of the oligonucleotide itself. Include non-targeting controls to differentiate these possibilities.
Poor Tissue Distribution
- Consider fluorescent labeling: Fluorescently labeled sdASO™ can help visualize tissue distribution and cellular uptake.
- Change administration route: Different routes lead to different distribution patterns. IV generally provides good distribution to liver, kidney, and other highly perfused organs. For CNS targets, IT administration may be necessary.
- Increase dose or frequency: Higher doses or multiple administrations may improve distribution to less accessible tissues.
- Special considerations for CNS: The blood-brain barrier limits distribution to CNS tissues from systemic administration. For CNS targets, consider intrathecal, intracerebroventricular, or intracerebral administration.
Storage and Additional Information
Storage Conditions
- Store lyophilized sdASO™ at -20°C upon receipt.
- Reconstituted stock solutions should be stored in single-use aliquots at -20°C.
- Avoid repeated freeze-thaw cycles, as they may affect sdASO™ stability and efficacy.
- Formulated sdASO™ solutions for in vivo administration should be prepared fresh when possible. If storage is necessary, keep at 4°C for no more than 24 hours.
Additional Considerations
- Duration of effect: Depending on the target and tissue, knockdown typically persists for 1-4 weeks following a single administration.
- Species considerations: While the basic principles apply across species, doses may need adjustment for larger animals.
- Biodistribution: sdASO™ distribution varies by tissue, with liver, kidney, and spleen typically showing the highest accumulation following systemic administration.
- Pharmacokinetics: sdASO™ typically have plasma half-lives of several hours to days, with tissue half-lives of days to weeks depending on the chemical modifications.
AUM Biotech's sdASO™ products are for research use only. Not for use in diagnostic or therapeutic procedures. All animal studies should be conducted in accordance with institutional animal care guidelines and with proper ethical approvals.
Ready to Advance Your In Vivo Research?
Our scientific team is available to help you design optimal sdASO™ solutions for your in vivo research needs. Contact us for personalized support or to request a quote for custom sdASO™ design.
Related Resources
Tissue Distribution Guide
Comprehensive guide to sdASO™ distribution in various tissues and administration routes.
View GuideFAQs
Find answers to frequently asked questions about in vivo applications of AUM Biotech's sdASO™ products.
View FAQs