**2. Adeno‐associated virus‐mediated gene transfer to neonate**

Among the numerous viral and nonviral vectors that have been developed to deliver genes of interest into target cells, adeno‐associated virus (AAV) vector has emerged as a particularly prom‐ ising tool for gene delivery, thanks to its safety (AAV is not pathogenic) and its ability to transduce nondividing cells [7–9]. We are now using several AAV vector serotypes (mainly 1–12), depend‐ ing on the target [10–13]. **Figure 1** shows the results after intravenous injection into neonatal

**Figure 1.** Systemic intravenous injection of AAV vectors into neonatal mice. (A) Approximately 5.0 × 1011 vector genomes (vg) of recombinant AAV vectors encoding the luciferase gene (AAV/Luc) (serotype 1, 8, 9) were injected into the external jugular vein of neonatal mice using a syringe with a 29‐G needle. Bioluminescent images of mice were obtained using a Xenogen IVIS imaging system 3 days and 2, 4, 8, 12 and 16 weeks after administration. Color scale bar indicates radiant efficiency (photons s−1 cm−2 steradian−1 per µW cm−2). (B) Radiant efficiency of serotype 1 (blue), 8 (red), and 9 (green) AAV vectors injected mice was quantified. (C) Approximately 5.0 × 1011 vg of AAV vectors encoding green fluorescent protein (serotype 1, 8, 9) were injected into the external jugular vein of neonatal mice. Sixteen weeks after injection, liver, heart and muscle were stained with anti‐GFP antibody.

mice of AAV vector serotypes 1, 8 and 9, harboring the luciferase gene. Expression of luciferase was detected within 3 days and continued for more than 16 weeks with no decrease in expres‐ sion. Serotype 9 mediated the highest expression during the observation period (**Figure 1A, B**). In addition, using an AAV vector encoding green fluorescent protein (GFP), we determined that the organs most efficiently transduced are the liver, heart and muscle (**Figure 1C**). Moreover, although transduction efficiency was not as high, the central nervous system (CNS) was also transduced after intravenous injection of AAV vector, which apparently passes through the blood‐brain barrier (BBB) [14] in neonatal mice [15]. Thus, a systemically administered AAV vec‐ tor was able to transduce several important target organs in neonatal mice, including the CNS, and mediate expression of a gene of interest for a prolonged period of time.