Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

Summary

The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice.

Abstract

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 10¹³ genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Introduction

The use of AAV vectors to treat a variety of spinal cord and CNS neurodegenerative disorders is becoming a well-accepted platform to effectively upregulate or silence the expression of gene(s) of interest. One of the key limitations to the more effective utilization of this technology to treat CNS/spinal cord disorders is the limited ability to deliver AAV vector(s) to the deep brain or spinal cord parenchyma in adult mammals.

It was demonstrated, for example, that the systemic delivery of AAV9 in adult rodents, cats, or non-human primates is only moderately effective at inducing transgene expression in neurons in the brain and spinal cord^1,2,3. The more effective intrathecal delivery of AAV9 vectors has also been shown to lead to only limited transgene expression in anatomically-defined pools of neurons. More specifically, it has been demonstrated that cisternal or lumbo-sacral intrathecal AAV9 delivery in non-human primates, pigs, or rodents leads to a high level of transgene expression in spinal α-motoneurons and segmental dorsal root ganglion neurons. However, minimal or no expression in spinal interneurons or ascending or descending axons in the white matter is seen^4,5,6,7. Collectively, these data show that a highly effective biological-anatomical barrier exists, which prevents the diffusion of intrathecally delivered AAV into deeper spinal parenchyma.

In a previous study using adult rats and pigs, a novel subpial vector delivery technique was developed⁸. Using this approach, highly potent and multi-segmental transgene expression was demonstrated after a single-bolus subpial AAV9 delivery. Intense GFP expression was consistently seen in neurons, glial cells, and descending/ascending axons through the injected spinal segments. This study demonstrated for the first time that the pia mater represents the primary barrier limiting effective AAV9 diffusion into the spinal parenchyma from the intrathecal space. While this previously developed technique and subpial injection device is relatively easy to use in large rodents (like rats) or adult pigs, the system is not suitable for use in small animals, such as adult mice. Because of the high number of available transgenic mouse models of a variety of neurodegenerative disorders, there is a clear need for the development of an effective spinal-parenchymal vector delivery technique in mice. The availability of such a technique would permit the study of the effect of specific gene silencing (e.g., using shRNA) or upregulation using cell-non-specific (e.g., cytomegalovirus-CMV or Ubiquitin) or cell-specific (e.g., synapsin or glial fibrillary acidic protein (GFAP)) promoters during early postnatal development or under diseased conditions.

Accordingly, in the present study, we have developed and validated a miniature subpial vector delivery system that can effectively be used in adult mice. Similarly, as in previous rat and pig studies, this work demonstrates potent transgene expression throughout the spinal parenchyma after a single-bolus subpial AAV9 delivery in mice. The simplicity of this approach, the very good tolerability of injected mice to subpial AAV9 delivery, and the high potency of transgene expression in the spinal parenchyma suggest that this technique can effectively be implemented in any laboratory setting and used in experiments targeting spinal gene expression.

Protocol

These studies were carried out under a protocol approved by the Institutional Animal Care and Use Committee of the University of California, San Diego and were in compliance with the Association for Assessment of Laboratory Animal Care guidelines for animal use. All studies were performed in such a manner as to minimize group size and animal suffering.

1. General Animal and Surgical Preparation

1. Before starting the surgical procedure, thaw the virus (AAV9-UBI-GFP; 5 µL aliquots)⁸. Prepare a 5% dextran (10,000 MW) solution by mixing dextran powder in distilled water. Mix the virus solution with 5% dextran solution 1:1 to a final dextran concentration of 2.5%.
   1. Store the virus solution on ice (4 °C).
2. Use adult C57BL/6J mice (male and female, 20-30 g). Anesthetize the mice using 5% isoflurane (in O₂, 1 L/min) and maintain them at 2-3% inhaled isoflurane (in O₂, 1 L/min) by nose cone during surgery, depending on the breathing rate and paw pinch response.
3. Shave the back of the animals with shaving clippers and clean the skin with 2% chlorhexidine.
4. In chronic recovery studies follow a strict sterile technique.
5. If lumbar subpial injections are to be performed, cut the skin overlaying the Th8-L1 vertebrae with a scalpel and detach the paravertebral muscle from Th10-12 spinal vertebrae using scissors.
   1. Mount the animal into a standard stereotaxic frame using mouse spinal clamps.
   2. Shave the both sides of the lamina of the Th10-12 vertebrae using a dental drill (drill bit: 0.9 mm, speed: 20,000 rpm) until cracks appear.
   3. Remove cracked bone fragments with forceps and expose the dorsal surface of the lumbar spinal cord.
   4. Cut open the dura about 1 cm using a 30 G stainless steel needle and forceps.
6. If cervical subpial injections are to be performed, incise the dorsal neck skin 1.5-2 cm using scissors and expose the C1-C2 segments.
   1. Remove the atlanto-occipital membrane of the cisterna magna using a 23G stainless steel needle and forceps.
   2. Clean the incision site of any tissue and bone debris using cotton swabs.
   3. Cut open the dura about 2-3 mm using a 30 G stainless steel needle and forceps.

2. Opening the Pial Membrane and Inserting the Subpial Needle for AAV9 Delivery

1. Mount the 34 G pia-penetrating needle into the Z-arm of an XYZ manipulator using a glass capillary holder (Figure 1A and B).
   NOTE: To manufacture the pia-penetrating needle, the original beveled tip of the 34G needle is sharpened using a glass capillary beveller with diamond abrasive plate - coarse (5.0 µm to 50 µm tip sizes) and grinding angle of 15 - 20°. The tip of the needle (1 mm length, measured from the tip) is then gently bent to about 90° (Figure 1B, left insert).
2. Using a surgical dissecting scope, set to 8-10 X magnification, penetrate the pia with the pia-penetrating needle by about 1 mm (Figure 1C) using the X-arm.
   1. Keep the angle of the penetrating needle to the tissue surface at 5-10°.
3. After the pia opening, remove the pia-penetrating needle horizontally from the subpial space (Figure 1 D) using the X-arm.
   NOTE: Remember the penetrated site by a landmark, such as a blood vessel.
4. Load a blunt 36G injection needle with AAV9-UBI-GFP virus using a 50-µL microsyringe connected to the injection needle with PE-10 or PE-20 tubing.
5. Mount the needle into the Z-arm of a second XYZ manipulator (Figure 1A and B) using a glass capillary holder (Figure 1B, right insert).
   NOTE: To manufacture the subpial AAV9 injection needle, the blunt tip of a 36 G needle is polished using a glass capillary beveller with diamond abrasive plate - coarse (5.0 µm to 50 µm tip sizes) to remove the sharp edges. The tip of the needle (2-3 mm length, measured from the tip) is then gently bent to about 90°. The pia-penetrating and subpial injection needles are inserted into 1-2 cm-long 20G slave stainless steel tubing (10 mm from the end of the needle) and glued with epoxy. The use of 20 G (0.91 mm-diameter) tubing is required for a secure attachment to the glass capillary holder.
6. By manipulating the X, Y, and Z arms of the second manipulator, position the tip of the AAV9 injection needle into the pia-penetrated site and then advance it about 2-3 mm into the subpial space through the previous pial membrane opening using the X-arm ( Figure 1E and F).
   NOTE: 1) The AAV9-UBI-GFP is prepared according to previously reported protocols^9,10, and the final titers are adjusted to 1.2 x 10¹³ genome copies per mL (gc/mL). 2) There is no need to mark the site of the pial opening because it is readily identifiable (Figure 1D).
7. Inject the AAV9-UBI-GFP (1.5, 3, or 5 µL) into the subpial space using a 50 µL microsyringe (see Table 1 for experimental groups).
8. Remove the injection needle from the subpial space after the AAV9-UBI-GFP injection is complete.
9. Close the muscle and skin using 4.0 monofilament suture and surgical clips.
   NOTE: There is no need to seal the open vertebra.
10. Allow the animals to recover on a heating pad.
11. For pain control inject Buprenorphine 0.05 mg/kg/sc  every 12 h for 2-3 days post-surgery.

3. Perfusion-fixation, Tissue Cryoprotection, and Immunofluorescence Staining

1. At a predetermined time point after the subpial AAV9 injections, deeply anesthetize the mice with euthanasia solution (see the Table of Materials, 0.3 mL) and transcardially perfuse them with 20 mL of heparinized saline followed by 20 mL of 4% paraformaldehyde in PBS.
2. Dissect the spinal cords and brains using a bone rongeur and post-fix them in 4% formaldehyde in PBS overnight at 4 °C.
3. Cryoprotect the spinal cords and brains with 30% sucrose in PBS for a minimum of 5-7 days.
4. Cut coronal, transverse, or longitudinal/horizontal frozen sections (30 µm-thick) on a cryostat and store them in PBS at 4 °C.

4. Immunofluorescence Staining of Spinal Cord and Brain Sections (See the Table of Materials)

1. Incubate free-floating sections in primary antibodies overnight.
2. After incubation with primary antibodies, wash the sections three times in PBS and incubate with fluorescence-conjugated donkey anti-rabbit, donkey anti-chicken, and donkey anti-goat secondary antibodies.
3. Mount the sections on microscopy slides, dry them at room temperature, and cover them with an anti-fade medium.
4. Capture images using epifluorescence fluorescence microscope (objectives: 10X, NA-0.3; 20X, NA-0.8; and 63X, NA-1.4).

Results

Potent Transgene Expression in Subpially AAV9-injected Segments:
The analysis of transgene (GFP) expression in spinal cord sections at 14 days after AAV9 delivery showed AAV9-dose dependent GFP expression throughout the spinal parenchyma. First, two bilateral 3 µL injections of AAV9-UBI-GFP injected into the upper lumbar subpial space were associated with the near-complete infection of the white and gray matter in the whole lumbar spinal cord, extending to the ...

Discussion

The current study describes a technique of subpial vector (AAV9) delivery in adult mice. As demonstrated in the accompanying video, this approach and technique can effectively be used, provided that the required instruments and pia-penetrating needle and subpial injection needle are properly manufactured, according to the established and tested specifications.

Critical Technical Variables in Performing a Consistent and Safe Subpial Injection in Mice:
As demonstrated, a s...

Materials

Name	Company	Catalog Number	Comments
C57BL/6J Mice	Jackson Labs	664
Lab Standard Stereotaxic for Mice	Harvard Apparatus	72-9568
Mouse Spinal Adaptor	Harvard Apparatus	72-4811
XYZ Manipulator	Stoelting	51604
Manual Infusion Pump	Stoelting	51218
34G Beveled Nanofill Needle	World Precision Instruments	NF34BV-2
36G Blunt Nanofill needle	World Precision Instruments	NF-36BL-2
Fluriso, Isoflurane	MWI Veterinary Supply	502017
Chlorhexidine Solution	MWI Veterinary Supply	501027
20G Stainless Steel Needle	Becton-Dickinson	305175
23G Stainless Steel Needle	Becton-Dickinson	305145
30G Stainless Steel Needle	Becton-Dickinson	305128
Cotton Tipped Applicator	MWI Veterinary Supply	27426
Glass Capillary Beveller	Narishige International	SM-25B
Slide Microscope Superfrost	Leica Microsystems	M80
50μl Microsyringe	Hamilton	81242
BD Intramedic PE-20 Tubing	Becton, Dickinson	427406
BD Intramedic PE-10 Tubing	Becton, Dickinson	427401
4-0 monofilament suture	VetOne	V1D397
Glass Capillary Beveller	Narishige	Pipet Micro Grinder EG-40
5 min Epoxy (Epoxy Clear)	Devcon	14310
Euthanasia Solution	MWI Veterinary Supply	11168
Heparin Inj 1000U/mL	MWI Veterinary Supply	54254
Paraformaldehyde	Sigma-Aldrich	158127
Sucrose	Sigma-Aldrich	S0389
Anti NeuN Antibody	EMD-Millipore	ABN78	Primary Rabbit Polyclonal Antibody, 1:1000
Anti-Choline Acetyltransferase (CHAT) Antibody	EMD-Millipore	AB144P	Primary Goat Polyclonal Antibody, 1:100
Anti GFP Antibody	Aves Labs	GFP-1020	Primary Chicken Polyclonal Antibody, 1:1000
Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594	ThermoFisher Scientific	A21207	Secondary Antibody, 1:1000
Donkey anti-Rabbit IgG Secondary Antibody, Alexa Fluor 680	ThermoFisher Scientific	A10043	Secondary Antibody, 1:1000
Donkey anti-Chicken IgY Secondary Antibody, Alexa Fluor 488	Jackson Immunoresearch Labs	703-545-155	Secondary Antibody, 1:1000
Donkey Anti-Goat IgG H&L (Alexa Fluor 647	Abcam	ab150131	Secondary Antibody, 1:1000
Slide Microscope Superfrost	Fisher Scientific	12-550-143
ProLong Gold Antifade Mountant	Fisher Scientific	P36930
Epifluorescence Microscope	Zeiss	Zeiss AxioImager M2
Fluorescence Confocal Microscope	Olympus	Olympus FV1000
Dextran	Polysciences, Inc	19411
AAV9-UBC-GFP	UCSD Viral Vector Core Laboratory