A number of different injection routes have been established for delivery of oligonucleotides into animals, such as systemic delivery through the tail vein (TV) or local delivery through intraperitoneal (IP), intranasal (IN), intramuscular (IM), intratracheal (ITr), intrathecal (ITh), intratumoral (ITu) or subcutaneous (SC) administration. Below are protocol guidelines for systemic and intraperitoneal administration of siRNA, the two most commonly used delivery routes in mice.

Systemic Delivery
Organs typically targeted by this method: liver, kidney, spleen, tumors, xenografts

Background
Systemic delivery includes the following administration routes in rodents and non-human primates:

•  Intravenous (IV)
•  Subcutaneous (Sub-Q)
•  High Pressure Tail Vein (HPTV)†
•  Low Pressure Tail Vein (LPTV)

Systemically delivered siRNAs go through first-pass metabolism in the liver. Hence, liver is the most commonly targeted organ using this approach.

Intravenous administration of a single dose of antisense oligonucleotides in rodents revealed elimination from the plasma that was biphasic in nature [1]. An initial, short half-life (~ 0.2 to 0.7 hours) due to distribution out of the plasma compartment was followed by a second, long half-life (up to 50 hours) reflecting elimination from the body. The half-life of distribution from plasma to tissue increased with dose in a stepwise manner. Urinary excretion was the major pathway of elimination from the body.

Subcutaneous dosing produced greater distribution of oligonucleotide into tissue and reduced excretion of the administered dose [2]. However, this advantage was offset by the greater material requirements of subcutaneous adminstration.

Recommended Buffers for Administering the siRNA

•  Sterile saline (0.9 % NaCl), or variants containing sugars such as mannitol or glucose (5–15%)
•  Sterile, phosphate buffered saline (PBS)
•  Ringer's solution: 147 mM NaCl, 4 mM KCl, 1.13 mM CaCl₂

Dosing Strategy/Protocol
Continuous infusions have been carried out in animal models for as long as 2 weeks; bolus injections have been given as often as three times a day. For effective mRNA knockdown, both dosage and dosing strategy may require adjustment based on routes of clearance from the target tissue. A majority of systemically delivered siRNA are excreted through the kidneys; thus, continuous infusion or multiple bolus injections might be an appropriate dosing strategy in order to maintain plasma levels. Tail vein administration is the preferred route for targeting tumor xenograft models (planted subcutaneously or via orthotopic implantation).

For systemic delivery, dried siRNA is diluted into the appropriate buffer at the chosen concentration. The siRNA can also be formulated with a delivery agent, if desired. Table 1 shows suggested dosages for a typical siRNA.

Dose (mg/kg)	siRNA Dose for a 25 g Mouse (nmol)	siRNA Concentration Needed for a 200 µl Injection ( µM )
1.0	1.93	9.6
5.0	9.63	48.1
10.0	19.25	96.3
15.0	28.88	144.4
20.0	38.50	192.5
50.0	96.25	481.3
Table 1. siRNA concentrations needed to reach commonly used siRNA doses in mice.

Caution: Sterility of all buffers, tubes, and syringes/catheters should be maintained. For injection of 200 µl into the tail vein, use of a 1 ml syringe and 27-gauge needle is recommended.

†In vivo hydrodynamic delivery of nucleic acids is covered by patents and patent applications of Mirus Bio Corporation, including U.S. Patents 6,627,616, 6,379,966 and 6,897,068 and related filings worldwide. Research and commercial uses by for-profit entities require a license--please see www.mirusbio.com for contact information.

Intraperitoneal Delivery
Organs typically targeted by this method: Organs and tumors in the abdominal region

Background
Intraperitoneal (IP) delivery introduces siRNA into the peritoneal cavity—the space between the parietal peritoneum, the membrane attached to the abdominal wall, and the visceral peritoneum, the membrane wrapped around the internal organs of the abdominal cavity. IP administration is the preferred route for siRNA delivery to most organs in the abdominal region, as it avoids sequestering of the oligonucleotide in the lung and liver. IP administration can be used to target tumors in the peritoneal cavity. It should be noted that nucleic acids do diffuse out of the peritoneal cavity to reach the blood and lymph circulation.

Recommended Buffers for Administering the siRNA

•  Sterile saline (0.9 % NaCl), or variants containing sugars such as mannitol or glucose (5–15%)
•  Sterile, phosphate buffered saline (PBS)
•  Ringer's solution: 147 mM NaCl, 4 mM KCl, 1.13 mM CaCl₂

Dosing Strategy/Protocol
Continuous infusion using an osmotic minipump, as well as bolus administration can be used for this delivery route. The usual volume of injection ranges from 100–400 µl. Suggested dosages are shown in Table 1.

For further information, please see our spreadsheet of published siRNA delivery routes and dosing strategies [Excel format].

References:

1. Phillips JA, Craig SJ, Bayley D, Christian RA, Geary R, Nicklin PL. (1997) Pharmacokinetics, metabolism, and elimination of a 20-mer phosphorothioate oligodeoxynucleotide (CGP 69846A) after intravenous and subcutaneous administration. Biochem Pharmacol. 54(6):657-68.
   
2. Raynaud FI, Orr RM, Goddard PM, Lacey HA, Lancashire H, Judson IR, Beck T, Bryan B, Cotter FE. (1997) Pharmacokinetics of G3139, a phosphorothioate oligodeoxynucleotide antisense to bcl-2, after intravenous administration or continuous subcutaneous infusion to mice. J Pharmacol Exp Ther. 281(1):420-7.