sdASO™ in vivo protocol

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)

1. 01

   Step 1: 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).

   Note: 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.

2. 02

   Step 2: 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 the target dose. AUM's own guidance is to test a dose range, 3-30 mg/kg in mice, to find the dose that silences best. Lower and higher doses may be needed, depending on the administration route and on how long activity has to be sustained. The dosing table below adds a starting dose and a maximum for each animal model.
   • 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.

   Important: 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.

3. 03

   Step 3: 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 tip: 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, intrathecal administration puts the oligonucleotide into the cerebrospinal fluid and so bypasses the blood-brain barrier rather than crossing it.

4. 04

   Step 4: 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. When knockdown is maximal varies with the target, the tissue, the dose and the administration route. Collect at more than one timepoint rather than at a single harvest.
   • 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)

   Note: The kinetics of knockdown vary by tissue and target. Multiple timepoints are recommended for initial studies to establish optimal analysis timepoints.

5. 05

   Step 5: 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.

   Analysis tip: 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 depends on the target, tissue, dose, and administration route.

Reference guidelines

The tables below provide guidelines for dosing and administration routes:

Optimization tips and best practices

Troubleshooting common issues

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.

• In vitro protocol Detailed protocol for using sdASO™ in cell culture applications.
• Tissue distribution guide sdASO™ distribution in various tissues and administration routes.
• In vivo studies on this site The in vivo studies this site records, each with its model, its dose and its own numbers.
• FAQs Find answers to frequently asked questions about in vivo applications of AUM BioTech's sdASO™ products.