Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

Summary

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

Abstract

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Introduction

The vascular system of the central nervous system (CNS) has evolved as a critical regulator of homeostasis, controlling traffic of molecules, supplying nutrients and getting rid of waste. This system is also the first line of defense from attacks of external pathogens, thanks to a dense distribution of tight junctions along the walls of the endothelial cells. These tight junctions make up one aspect of the blood brain barrier (BBB). While the BBB allows the transport of molecules required to fulfill nutrient and energy demands (e.g., ions, glucose), it also selectively limits the passage of pathogens as well as toxic chemicals^1,

Protocol

All in vivo procedures were performed under Biogen Institutional Animal Use and Care Committee (IACUC) approved protocols which follow the guidelines set forth by the United States National Institutes of Health guide for the care and use of laboratory animals.

1. Material and instrument preparation

1. Prepare the special guide cannulas.
   1. Use a rotary tool with cut-off wheel (or a sharp saw) to cut off the two ends of a 19 G needle, resulting in a ~1.5−2 cm long guide cannula (Figure 1Aiii). Use the grinding wheel of the rotary tool to smooth the two ends.
      NOTE:....

Results

Using the method described here, we injected two groups of adult female rats (250−300 g; n = 10/group) with either a single bolus of phosphate-buffered saline PBS or 300 µg of ASO targeting the long non-coding (linc) RNA Malat1; in our lab we routinely use the Malat1 ASO as a tool compound, because Malat1 is expressed ubiquitously and at high levels in all tissues¹⁴, including brain and spinal cord. The Malat1 ASO works via an RNaseH1-mediated mechanism

Discussion

The present article shows a powerful method to deliver therapeutic agents directly into the rat CNS. In theory, a similar technique can be also performed in mice, though due to the smaller size, the method can be more challenging. Therefore, our group performs intracerebroventricular (ICV) injections in mice for CNS drug delivery, which reach the same goals through a different route of administration. That method has been described in another study¹⁶.

The advantage of t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3M Steri-Drape Small Drape with Adhesive Aperture	3M		1020
70% ethanol	Decon Laboratories, Inc		8416-160Z
Alcohol swab sticks	Dynarex		NO 1204
BD General Use Syringes 1 mL Luer-Lok tip	BD	1ml TB Luer-Lok tip	BD 302830
BD Intramedic PE Tubing	BD	Polyethylene tubing PE50 Diameter 0.023 in	BD 427400 (10ft, Fischer Scientific 22-204008) or 427401 (100ft, Fischer Scientific 14-170-12P)
BD Intramedic PE Tubing	BD	Polyethylene tubing PE10 Diameter 0.011 in	BD 427410 (10ft, Fischer Scientific 14-170-11B) or 4274011 (100ft, Fischer Scientific 14-170-12B)
BD Intramedic PE Tubing Adapters	BD	23 gauge intramedic luer stub adaper	BD 427565 or Fisher Scientific 14-826-19E
BD PrecisionGlide Single-use Needles 30G	BD		BD 305128
Buprenorphine Sustained Release-lab	ZooPharm	Prescription required
Ethylene oxide sterilizer	Andersen Sterilizer INC.	AN 74i, gas sterilizer	AN 74i
Guide cannula	BD	19G x 1 WT (1.1 mm x 25mm) needle	BD 305186
Hamilton syringe 100ul	Hamilton company	Hamilton syringe 100ul
Hot bead Sterilizer	Fine Science Tools		STERILIZER MODELNO FST 250
Ophthalmic ointment	Dechra veterranery product		17033-211-38
Pocket Pro Pet Trimmer	Braintree Scientific		CLP-9931 B
Povidone scrub	PDI		S48050
Saline	Baxter	Sodium Chloride 0.9% Intravenous Infusion BP 50ml	FE1306G
Scalpel	Feather	disposable scalpel	No. 10
Small animal heating pad	K&H Manufacturing		Model # 1060
Stylet Wire	McMaster-Carr	1749T14	LH-36233780
Surgery Towel drape	Dynarex		4410
Surgical scissors and forceps	FST and Fisher Scientific
Sutures	Ethicon		4-0 or 5-0
Tool to make the Guide cannular	Grainger	Rotary tool (Dremel)	14H446 (Mfr: EZ456)
		EZ lock cut off Wheel	1PKX5 (Mfr: 3000-1/24)
		Grinding Wheel, Aluminum Oxide	38EY44 (Mfr: EZ541GR)
		EZ lock Mandrel	1PKX8 (Mfr: EZ402-01)
		Diamond wheel floor Tile	3DRN4 (Mfr: EZ545)
Alternative source for pre-made and sterilized materials for this procedure
Dosing catheter system	SAI Infusion Systems		RIDC-01
Guide cannula	SAI Infusion Systems		RIDC-GCA
Internal Catheters	SAI Infusion Systems		RIDC-INC
Stylet Wire	SAI Infusion Systems		RIDC-STY