**5. Application of AAV vectors to CNS disorders**

**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,

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

expression of GFP was analyzed using fluorescent microscopy at 5 weeks post administration.

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

112 Gene Therapy - Principles and Challenges

if one wants to also transduce the peripheral nervous system.

As summarized above, AAV vectors are an effective means of delivering genes into the CNS, thanks to their ability to transduce post-mitotic neurons and mediate efficient and stable transduction. Indeed, the utility of directly delivered AAVs has been demonstrated in numerous preclinical studies, and they are currently being used in clinical trials of treatments for Alzheimer's disease [18, 19], Parkinson's disease [20, 21], Canavan's disease [22], and Batten's disease [23, 24], among others. We also evaluated the utility of brain directed [25], intracerebroventricular [16], intrathecal [17], and intravenous neonatal administration [26] of AAV vectors for the treatment of metachromatic leukodystrophy (MLD), an inherited lysosomal storage disease with severe neurological symptoms. When we injected AAV9 vectors expressing human arylsulfatase A (AAV9/ASA) into the jugular vein of newborn MLD model mice, efficient ASA expression was detected throughout the entire brain (Fig. 7A) and peripheral nervous system (Fig. 7C), suppressing the accumulation of sulfatides in both CNS (Fig. 7B) and PNS (Fig. 7D). Moreover, the treated mice showed a greater ability to traverse narrow balance beams than untreated mice [26]. These data clearly demonstrate that MLD model mice can be effectively treated through systemic administration of AAV9/ASA vector to neonates. Thus, neonatal gene therapy is one approach with the potential to overcome the limitation imposed by the BBB on treating genetic disorders of the CNS. Other advantages of systemic gene transfer to neonates with genetic disease over treatment of adults are as follows: (1) because the immune system is immature, recipients are immunologically tolerant of the vector; (2) it may prevent early onset of genetic diseases; (3) neonates can be effectively treated with a smaller amount of vectors than adults; and (4) nearly all organs are efficiently trans‐ duced. Systemic neonatal gene therapy thus appears to be a promising method for treating systemic genetic diseases with neurological symptoms.

**Figure 7.** Correction of sulfatide storage by neonatal systemic injection of AAV9/ASA vectors. After injection of AAV9/ASA into the jugular vein of newborn MLD mice, ASA expression in the brain (A) and spinal cord (C) was ana‐ lyzed by immunohistochemistry using an anti-ASA antibody at 15 months after injection. Correction of sulfatide stor‐ age in the brain (B) and spinal cord (D) was analyzed by alcian blue staining.
