**3. Local gene delivery to the CNS using AAV vectors**

Brain-directed local injection of AAV vectors is a straightforward approach to gene transfer to the CNS. We compared the transduction efficiency of several AAV vector serotypes encoding the luciferase gene (AAV/Luc) after intracranial injection in mice. Fig. 1 shows the resultant transduction efficiencies determined using an in vivo imaging system (IVIS). Efficient trans‐ duction was achieved using the AAV9/Luc or AAV10/Luc vectors compared to the AAV1/Luc or AAV8/Luc vectors, and sustained expression was detected for at least 6 months after injection. Notably, however, following injection of a small amount of AAV1/Luc or AAV9/Luc vectors (2 µl) into the striatum, expression of the transgene was detected in the liver after 2 weeks of injection (Fig. 1A). Therefore, although expression of the transgene was absent at 6 months after injection, one must be aware of the potential for the occurrence of unexpected transduction following directed local injection of an AAV vector.

Fig. 2 shows another comparison of transduction efficiency after local administration to the CNS, this time using AAV vectors encoding GFP (AAV/GFP). As expected, the AAV9/GFP vector exhibited the greatest ability to transduce neuronal cells 2 weeks after injection. Surprisingly, however, nearly the same high transduction efficiency was detected 2 months after injection of the AAV2/GFP vector. Although a long time is needed to achieve strong expression with AAV2, since there is no limitation for the patient, the use of AAV2 is one option for highly efficient CNS transduction.

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

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 of direct injection [10, 11].
