Elsevier

Journal of Controlled Release

Volume 196, 28 December 2014, Pages 71-78
Journal of Controlled Release

Intra-arterial delivery of AAV vectors to the mouse brain after mannitol mediated blood brain barrier disruption

https://doi.org/10.1016/j.jconrel.2014.09.018Get rights and content

Abstract

The delivery of therapeutics to neural tissue is greatly hindered by the blood brain barrier (BBB). Direct local delivery via diffusive release from degradable implants or direct intra-cerebral injection can bypass the BBB and obtain high concentrations of the therapeutic in the targeted tissue, however the total volume of tissue that can be treated using these techniques is limited. One treatment modality that can potentially access large volumes of neural tissue in a single treatment is intra-arterial (IA) injection after osmotic blood brain barrier disruption. In this technique, the therapeutic of interest is injected directly into the arteries that feed the target tissue after the blood brain barrier has been disrupted by exposure to a hyperosmolar mannitol solution, permitting the transluminal transport of the therapy. In this work we used contrast enhanced magnetic resonance imaging (MRI) studies of IA injections in mice to establish parameters that allow for extensive and reproducible BBB disruption. We found that the volume but not the flow rate of the mannitol injection has a significant effect on the degree of disruption. To determine whether the degree of disruption that we observed with this method was sufficient for delivery of nanoscale therapeutics, we performed IA injections of an adeno-associated viral vector containing the CLN2 gene (AAVrh.10CLN2), which is mutated in the lysosomal storage disorder Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL). We demonstrated that IA injection of AAVrh.10CLN2 after BBB disruption can achieve widespread transgene production in the mouse brain after a single administration. Further, we showed that there exists a minimum threshold of BBB disruption necessary to permit the AAV.rh10 vector to pass into the brain parenchyma from the vascular system. These results suggest that IA administration may be used to obtain widespread delivery of nanoscale therapeutics throughout the murine brain after a single administration.

Introduction

The brain is a notoriously difficult organ to access therapeutically due to the presence of the blood brain barrier (BBB). The BBB is a complex physical barrier of specialized endothelial and astrocytic cells that selectively prevents many molecules from passing from the blood to the brain parenchyma [1]. Since many drugs do not cross the BBB efficiently, achieving therapeutic concentrations of drug in the target tissue after systemic delivery can often require toxic doses, and many promising therapies for disorders of the central nervous system (CNS) fail due to inadequate delivery.

Direct delivery of therapy to the CNS through intra-parenchymal injection or controlled release implants (thereby physically bypassing the BBB) can obtain high local concentrations of the delivered agent, but these techniques are limited by the spatial distribution that can be obtained and require opening of the skull and meninges.

One technique that offers the potential to access large volumes of brain without performing open brain surgery is intra-arterial (IA) injection. In this technique a solution containing the agent of interest is injected directly into the arteries that supply the target tissue through an endovascular catheter [2], [3], [4], [5], [6], [7]. However, IA delivery to the CNS is still limited by the BBB. The most commonly used method of permeabilizing the barrier to allow transluminal transport of injected agents is mannitol mediated osmotic disruption which has been widely used in preclinical and clinical studies [3], [4], [8], [9], [10]. To date, most preclinical studies have been performed in larger animals due to the difficulty associated with surgical positioning of the endovascular catheter and as a result little data exist on IA injection in mice.

The large number of murine genetic models of CNS disorders would make the ability to reproducibly perform successful IA injections in mice of great value to the drug development community. Our goal in this work was to establish the injection parameters for IA delivery of therapeutic constructs to the brain after mannitol mediated BBB disruption in the mouse. We first examined the effects of IA mannitol injection flow rate and volume on the degree of BBB disruption using dynamic contrast enhanced magnetic resonance imaging (DCEMRI). We surgically implanted custom microcatheters in the internal carotid artery (ICA) of anesthetized mice and performed IA injections of the paramagnetic MRI contrast agent gadopentate dimeglumine (Gd-DTPA) while continually acquiring MR images 2 min after disrupting the BBB with an IA injection of 25 wt.%/vol. mannitol solution.

Next we applied these findings to the in vivo IA delivery of a rhesus macaque derived adeno-associated viral (AAV) vector to the brain to determine if the degree of disruption we observed would be sufficient for delivery of a relatively large nano-scale therapeutic construct. We performed IA injections of AAVrh.10CLN2, an AAV vector encoding the human CLN2 gene which is the gene of interest in Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL). The systemic nature of LINCL makes it an attractive potential target for IA delivery.

We found that the volume of the mannitol injection but not the flow rate had a significant effect on the degree of BBB disruption. Further we demonstrate that at high mannitol doses the BBB disruption is sufficient to allow transvascular delivery of AAV as evidenced by high levels of the transgene product which localized to neurons five weeks after treatment. We also show that the degree of BBB disruption seen on DCE-MRI scans is predictive of eventual viral particle deposition within the brain.

Section snippets

Endovascular microcatheters

Custom polyimide endovascular microcatheters were fabricated as previously described [11]. Briefly, 169 μm outer diameter monolumen polyimide tubing (MicroLumen, Tampa, FL) was cut to 10 cm lengths and one end was secured in a polypropylene injection hub (SmallParts Inc.) using two-part epoxy (Miller Stephenson, Danbury, CT). The adhesive was then cured for two hours at 80 °C. Next, a 13 cm length of 7-0 monofilament suture was passed into the lumen of the microcatheter to prevent the tubing from

DCE-MRI studies of mannitol mediated BBB disruption in the mouse

To assess BBB disruption resulting from IA infusion of mannitol, we measured contrast enhanced MRI signals in mouse brain regions accessed by IA delivery as a function of time in regions supplied by the anterior choroidal, middle cerebral, and anterior cerebral arteries (AChA, MCA, ACA respectively) (Fig. 1).

When compared to baseline images, Gd-DTPA introduced into the arteries initially caused a darkening of the vessels due to water T2 nuclear relaxation time effects [22]. The contrast agent

Discussion

By performing DCE-MRI after BBB disruption we were able to quantify the impact of different IA mannitol injection conditions on the extent of disruption in different territories of the brain. We found that increasing the dose of mannitol resulted in increased BBB disruption. This was true for measurements over the whole hemisphere and in individual arterial territories (Fig. 2).

Many current clinical IA mannitol delivery protocols inject 25 wt.%/vol. mannitol at the highest flow rate that

Conclusions

We showed that DCE-MRI is a powerful technique for quantifying the extent and pattern of BBB disruption after IA injection of a hyperosmotic mannitol solution. We found that the volume of injected mannitol is the most significant factor in BBB disruption, and that the degree of disruption is based on the order of arterial branching from the ICA. The flow rate of mannitol injection is not significant for determining disruption provided it is slow enough to allow sufficient contact time (>~ 30 s)

Acknowledgments

This investigation was supported in part by the National Institute of Neurological Disorders and Stroke under a Ruth L. Kirschstein National Research Service Award F32NS073397 (to CPF) and U01NS047458 (to RGC).

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    These authors contributed equally to this work.

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