Awake intracerebroventricular delivery and safety assessment of oligonucleotides in a large animal model

Abstract

Oligonucleotide therapeutics offer great promise in the treatment of previously untreatable neurodegenerative disorders; however, there are some challenges to overcome in pre-clinical studies. (1) They carry a well-established dose-related acute neurotoxicity at the time of administration. (2) Repeated administration into the cerebrospinal fluid may be required for long-term therapeutic effect. Modifying oligonucleotide formulation has been postulated to prevent acute toxicity, but a sensitive and quantitative way to track seizure activity in pre-clinical studies is lacking. The use of intracerebroventricular (i.c.v.) catheters offers a solution for repeated dosing; however, fixation techniques in large animal models are not standardized and are not reliable. Here we describe a novel surgical technique in a sheep model for i.c.v. delivery of neurotherapeutics based on the fixation of the i.c.v. catheter with a 3D-printed anchorage system composed of plastic and ceramic parts, compatible with magnetic resonance imaging, computed tomography, and electroencephalography (EEG). Our technique allowed tracking electrical brain activity in awake animals via EEG and video recording during and for the 24-h period after administration of a novel oligonucleotide in sheep. Its anchoring efficiency was demonstrated for at least 2 months and will be tested for up to a year in ongoing studies.

Keywords: CNS drug administration; CSF drug delivery; Ommaya reservoir; i.c.v. catheter; i.c.v. injection; large animal model; oligonucleotide safety assessment; siRNA.

Graphical abstract

Figure 1

Prototypes and illustrative schematic models (A) The top images represent the dorsal view of the disc prototype. The five openings on the sides are for fixing with ceramic screws in the animal’s skull. The groove in the middle of the device is where the intracerebral catheter will be seated. The bottom images represent the ventral view of the prototype. It is curved to accommodate the shape of the skull. (B) The images represent different angles of the "fixation table" prototype. This fixation table prototype is fitted over the disc prototype over the groove to secure the catheter in place. (C) Lateral view of an assembly of the disc and table prototypes with the passage of the catheter. The other end of the catheter is connected to the Ommaya reservoir. (D) Top diagonal view of the same assembly described in (C). (E) Side view of a prototype use model in an ovine cranium. The prototype disc is fixed on the frontal bone of the skull of the ovine models, and the Ommaya reservoir is attached on the occipital bone. (F) Diagonal back view of the same assembly as the model described in (E).

Figure 2

Lateral intraoperative X-ray images from intracerebroventricular (i.c.v.) catheter placement and reservoir replacement (A) Confirmation of a good catheter placement and i.c.v. targeting. The arrow indicates the catheter. The big arrowhead indicates the ventricular system being filled by contrast. The small arrowheads indicate the catheter and the catheter tract. (B) Representation of a mistargeting. The ruler indicates 6-mm deviation of the catheter tip in relation to the i.c.v. The deeper position of the catheter tip was detected during surgery and corrected. The arrow indicates the catheter tip. The big arrowhead indicates the targeted lateral ventricular system being filled with contrast from reflux in the catheter track. The small arrowheads indicate the catheter and the catheter tract. (C) Correction of the catheter position in relation to the previous image. The arrow indicates the catheter tip. The big arrowhead indicates the ventricular system i.c.v. targeted. The small arrowheads indicate the catheter and the catheter tract.

Figure 3

Electroencephalography electrodes channels (A) Illustrative representation of a sheep’s skull with the location of channels chosen for placement of electroencephalography (EEG) electrodes. (B) Demonstration of electrode placement (Ambu Subdermal Corkscrews – 47″) on a seated sheep for oligonucleotide infusion. (C) After reversal of sedation, still with the EEG electrodes attached to the scalp, the animal is kept with the trackIt device close to the body with an adaptation of a prolapse harness for sheep.

Figure 4

Electroencephalography readouts (A) Representation of a normal electroencephalography (EEG) reading. The scale bar represents 1 s of reading. (B) Detection of EEG abnormalities concomitant with oligonucleotide infusion. The black arrows indicate changes in the wave pattern. The peaks indicated by the arrows are consistent with seizure activity. (C) Detection of EEG abnormalities concomitant with oligonucleotide infusion. The black arrow indicates changes in the wave pattern. The dashed red line indicates the onset of tonic-clonic movements after the electrical peak indicated by the arrow, consistent with seizure activity.

Figure 5

Verification of the viability and functionality of the catheter 60 days after implantation (A) Sagittal view of magnetic resonance imaging indicating the location of the catheter. The white arrow indicates the catheter track. The white arrowhead indicates the lateral intracerebral ventricle. (B) Coronal view of magnetic resonance imaging indicating the location of the catheter. The white arrow indicates the catheter track. The white arrowhead indicates the left lateral intracerebral ventricle. (C) Lateral X-ray image of the animal’s skull with pre-injection of iodinated contrast. The black arrow indicates the exact location of the catheter tip. The round-headed black arrow indicates the reservoir. The small arrowheads indicate the catheter and the catheter tract. (D) Lateral image taken immediately after administration of iodinated contrast. The black arrow indicates the exact location of the catheter tip. The big arrowhead indicates the ventricular system being filled by contrast. The small arrowheads indicate the catheter and the catheter tract.

Figure 6

Lateral intraoperative X-ray images from intracerebroventricular catheter placement and reservoir replacement (A) X-ray single shot showing the broken catheter at the junction with the reservoir. The black arrow indicates the rupture between catheter and reservoir. The small arrowheads indicate the catheter and the catheter tract. (B) X-ray single shot after reservoir replacement and contrast administration. The arrowhead indicates the ventricular system being filled by contrast. The small arrowheads indicate the catheter and the catheter tract.

Figure 7

Placement and fixation of the prototype (A) Longitudinal incision and exposure of the skull incision location is based on neuro-navigation MRI coordinates. (B) Positioning of the disc prototype over the exposed skull, with the center hole overlapping the craniotomy location determined by neuro-navigation. (C) The skull underneath the grooves of the prototype is manually drilled to create threaded holes for the placement of ceramic screws. (D) The holes are made sequentially in a star pattern to prevent unwanted movement of the disc prototype. For each screw attached, the center hole of the prototype is verified via neuro-navigation to ensure proper positioning for the craniotomy. Representation of the fixation of the first ceramic screw. (E) Representation of the fixation of the five ceramic screws. (F) After the prototype is properly fixed to the skull, the craniotomy is performed in the center of the prototype following the predetermined angle by neuro-navigation to access the intracerebral ventricle. The black arrow indicates the craniotomy.

Figure 8

Intraoperative lateral X-ray images from intracerebroventricular catheter and reservoir placement (A) Animal positioned in the stereotactic with the needle-catheter system inserted into the left lateral ventricle. The black arrow indicates the bevel of the needle. The black reflection at the top of the image is the guide arm of the neuro-navigation equipment. (B) Single shot subsequent to (A), after contrast injection. The arrowhead indicates the ventricular system being filled by contrast. (C) Single shot after removal of the needle from the catheter lumen and injection of contrast to confirm catheter location. The square-headed arrow indicates the catheter. (D) Confirmation of the location after removing the guide arm from the neuro-navigation equipment. The arrowhead indicates the ventricular system being filled by contrast. (D.1) Dorsal view of the surgical field for injection of iodinated contrast for verification of catheter placement. (E) Implantation of the subcutaneous reservoir connected to the catheter. The round-headed arrow indicates the reservoir. (E.1) Dorsal view of the surgical field after implantation and fixation of the Ommaya subcutaneous reservoir. (F) Confirmation of the location of the catheter before closing the incision, via injection of iodinate contrast through the reservoir. The arrowhead indicates the ventricular system being filled with contrast, indicative of good targeting. (F.1) Dorsal view of the surgical field for injection of contrast through the Ommaya reservoir for confirmation of catheter placement.