**4. Systemic gene delivery to CNS using AAV vectors**

Although in some cases axonal transport may be useful for widespread transduction with AAV vectors, most often local injection of AAV vectors provides transgene expression only to limited regions in the CNS. Consequently, to obtain global transduction of the CNS, multiple intracerebral injections are needed. But this strategy is invasive, and safety becomes a problem. To overcome this problem, an ideal approach for efficient and safe transduction to CNS is systemic administration.

**Figure 2.** Brain-directed injection of AAV vectors encoding green fluorescent protein gene (AAV/GFP). Approximately 2.0 x 1010 vg of AAV/GFP vectors (serotypes 1, 2, 5, 8, 9, and 10) were injected into the right striatum over a period of 5 min using a Hamilton syringe with a 33-G blunt-tip needle. Expression of GFP was analyzed using fluorescent micro‐ scopy at 2 weeks (A) and 2 months (B) post administration.

#### **4.1. Systemic administration of AAV vectors for gene transfer to CNS**

#### *4.1.1. Systemic administration of AAV vectors during the neonatal period*

Finally, Fig. 3 shows results obtained with direct intracranial injection of AAV1/GFP vectors into the hippocampus (CA3). Although we injected AAV1/GFP vectors into the right hippo‐ campus (CA3), GFP expression was detected on both sides of the brain, indicating that GFP is efficiently transported to the left side through long axons. This axonal transport is an advantage

measurement of AAV/Luc transduction *in vivo* in the brain area 2 weeks and 6 months after injection.

**Figure 1.** Brain-directed injection of AAV vectors encoding the luciferase gene (AAV/Luc). (A) Approximately 2.0 x 1010 vector genomes (vg) of recombinant AAV/Luc vectors (serotypes 1, 8, 9, and 10) were injected into the right stria‐ tum over a period of 5 min using a Hamilton syringe with a 33-G blunt-tip needle. Bioluminescent images of mice were obtained using a Xenogen IVIS imaging system at 2 weeks and 6 months post administration. (B) Comparative

Although in some cases axonal transport may be useful for widespread transduction with AAV vectors, most often local injection of AAV vectors provides transgene expression only to limited regions in the CNS. Consequently, to obtain global transduction of the CNS, multiple intracerebral injections are needed. But this strategy is invasive, and safety becomes a problem. To overcome this problem, an ideal approach for efficient and safe transduction to CNS is

**4. Systemic gene delivery to CNS using AAV vectors**

of direct injection [10, 11].

108 Gene Therapy - Principles and Challenges

systemic administration.

Systemic administration of AAV vectors is a promising approach for widespread organ transduction, though the BBB is an obstacle to the transduction of the CNS. To overcome this problem, one possibility is to administer the vector during the neonatal period, when the BBB is immature. We injected AAV/GFP vectors (serotypes 1, 8, 9, and 10: 1.5 x 1011 vg each) into the jugular veins of neonatal mice and then used diaminobenzidine (DAB) staining to examine GFP expression. GFP signals were detected throughout the entire brain after injection of any of these serotypes. Efficient gene transfer was obtained by AAV9/GFP or AAV10/GFP vector injection (Fig. 4A). Fig. 4B shows immunohistochemical staining of GFP in the brain by systemic neonatal injection of AAV9/GFP vectors. GFP expression was detected throughout the brain, including the olfactory bulb, cerebral cortex, hippocampus, and brainstem, and the spinal cord was also transduced efficiently. However, after the use of the AAV8/GFP vector, widespread transduction in the brain was detected 2 weeks after injection. Moreover, global

**Figure 3.** Brain-directed injection of AAV1/GFP vectors into hippocampus (CA3). The CA3 regions of the hippocam‐ pus of 7-month-old mice were injected with AAV1/GFP vectors (8.0 x 109 vg) and examined 5 months later. Using a fluorescent microscope, slices of the hippocampal regions were analyzed for GFP expression.

expression of GFP was sustained for at least 18 months (Fig. 4C). Immunohistochemical staining revealed the presence of GFP within GFAP-positive astrocytes, NeuN-positive neurons, and Calbindin-positive Purkinje cells [6]. These findings suggest that systemic neonatal administration of AAV is an effective means of delivering transgenes to target neuronal systems.

#### *4.1.2. Systemic administration of AAV vectors after the neonatal period*

It is our experience that AAV vectors are able to pass through the BBB for at least 2 weeks after birth, but within 6 weeks, all AAV vectors lose the ability to cross the BBB [6]. Therefore, to transduce the CNS of adult mice, double-stranded (or self-complementary) AAV vectors (dsAAV) must be used [12]. When we injected single-stranded (ss) AAV9 or dsAAV9 vectors encoding GFP into the tail veins of 8-week-old mice and assessed GFP expression immuno‐ histochemically, minimal expression was detected in mice administered ssAAV9, whereas efficient GFP expression was achieved throughout the entire brain using dsAAV9 (Fig. 5). Thus, systemic administration of the dsAAV9 vector appears to be an effective means of transducing the CNS in adult mice. It was demonstrated that combined injection of AAV vectors with mannitol [13, 14] or use of ultrasound-targeted microbubble destruction [15] enhances gene expression in the brain after systemic injection of AAV vectors. Therefore, to improve gene delivery in the brain, systemic administration of the dsAAV9 vector, along with these strategies, may be a powerful tool for transduction to the CNS.

expression of GFP was sustained for at least 18 months (Fig. 4C). Immunohistochemical staining revealed the presence of GFP within GFAP-positive astrocytes, NeuN-positive neurons, and Calbindin-positive Purkinje cells [6]. These findings suggest that systemic neonatal administration of AAV is an effective means of delivering transgenes to target

**Figure 3.** Brain-directed injection of AAV1/GFP vectors into hippocampus (CA3). The CA3 regions of the hippocam‐

vg) and examined 5 months later. Using a

It is our experience that AAV vectors are able to pass through the BBB for at least 2 weeks after birth, but within 6 weeks, all AAV vectors lose the ability to cross the BBB [6]. Therefore, to transduce the CNS of adult mice, double-stranded (or self-complementary) AAV vectors (dsAAV) must be used [12]. When we injected single-stranded (ss) AAV9 or dsAAV9 vectors encoding GFP into the tail veins of 8-week-old mice and assessed GFP expression immuno‐ histochemically, minimal expression was detected in mice administered ssAAV9, whereas efficient GFP expression was achieved throughout the entire brain using dsAAV9 (Fig. 5). Thus, systemic administration of the dsAAV9 vector appears to be an effective means of transducing the CNS in adult mice. It was demonstrated that combined injection of AAV vectors with mannitol [13, 14] or use of ultrasound-targeted microbubble destruction [15] enhances gene expression in the brain after systemic injection of AAV vectors. Therefore, to improve gene delivery in the brain, systemic administration of the dsAAV9 vector, along with

*4.1.2. Systemic administration of AAV vectors after the neonatal period*

pus of 7-month-old mice were injected with AAV1/GFP vectors (8.0 x 109

fluorescent microscope, slices of the hippocampal regions were analyzed for GFP expression.

these strategies, may be a powerful tool for transduction to the CNS.

neuronal systems.

110 Gene Therapy - Principles and Challenges

OB, olfactory bulb; CC, cerebral cortex; Hip, hippocampus; PC, Purkinje cells in the cerebellum; BS, brain stem; SC, spinal cord.

**Figure 4.** Direct comparison of AAV serotypes to transduce CNS by neonatal systemic injection. (A) After serotype-1, -8, -9, or -10 AAV/GFP vectors were intravenously injected into neonatal C57BL/6 mice, cerebral GFP expression was analyzed by DAB staining 4 weeks after injection. Representative brain images showing immunohistochemistry using an anti-GFP antibody after AAV9/GFP (B) and AAV8/GFP (C) injection.

**Figure 5.** Immunohistochemical staining of brain sections of adult mice following systemic injection of ssAAV9/GFP or dsAAV9/GFP vectors. After 7.0 x 1012 vg of AAV9/GFP vectors were injected via tail veins of adult (7-week-old) mice, expression of GFP was analyzed using fluorescent microscopy at 5 weeks post administration.

#### **4.2. Intracerebroventricular and intrathecal injection of AAV vectors**

Another strategy for achieving global gene transfer into the CNS through systemic adminis‐ tration is vector delivery into the cerebrospinal fluid (CSF). There are two approaches to delivering an AAV vector into the CSF: intracerebroventricular injection and intrathecal injection. To evaluate the feasibility of intracerebroventricular injection, AAV1/GFP vectors were injected into the right lateral ventricle. Following the injection, GFP expression was broadly distributed in the choroid plexus and ependymal cells throughout the cerebral ventricles (Fig. 6A). Coronal brain sections revealed widespread diffusion of AAV1 from the injection site to the contralateral, anterior lateral and third ventricles, as well as the fourth ventricles via the cerebral aqueduct [16]. GFP expression was mainly confined to the choroid plexus and ependymal cells, with little or no detection of GFP in the brain parenchyma or spinal cord. Similarly, when we administered the AAV1/GFP vector intrathecally, GFP expression was broadly distributed throughout the brain (Fig. 6B). In addition, large numbers of nerve fibers in the dorsal spinal cord and the neuronal cell bodies in the dorsal root ganglia were also efficiently transduced [17]. Thus, it can be concluded that both intracerebroventric‐ ular and intrathecal injection of AAV vectors are useful for transduction of the CNS, especially if one wants to also transduce the peripheral nervous system.

**Figure 6.** Expression of GFP in the brain after intracerebroventricular or intrathecal injection of AAV vectors. (A) After injection of AAV1/GFP vectors into the right lateral ventricle, GFP expression in the brain was analyzed by fluores‐ cence microscopy (upper panel) or immunostaining with DAB staining (lower panel). (B) AAV1/GFP vectors were in‐ jected into the posterior cistern of 8-week-old mice and the brains were examined 8 weeks after injection. GFP expression was monitored by fluorescence microscopy.
