**3. Advantages of neonatal gene therapy**

Systemic gene transfer to neonates has several advantages over treatment of the adults (**Table 1**). First, as mentioned above, neonatal gene therapy has the potential to overcome the limitation imposed by the BBB on treating genetic disorders of the CNS. Because the BBB is developmentally immature during the perinatal period, AAV‐mediated neonatal gene therapy is a highly promising strategy for treating genetic neurological diseases. Second, because the immune system is immature, neonates are immunologically tolerant of the transgene and/or viral vector [16–18]. Immune rejection of the transgene product by neutralizing antibodies is a severe problem for gene therapy in adults. Third, treatment administered soon after birth may enable prevention of early‐onset genetic disease. Finally, neonates can be effec‐ tively treated with a smaller amount of viral vector than adults. Using smaller amounts of viral vector is superior with respect to both safety and cost. Taken together, these advan‐ tages make systemic neonatal gene therapy a promising method for treating systemic genetic diseases.


disorders [1–6]. However, although neonatal gene therapies have several advantages over similar therapies used in adult patients, there is as yet no clinical protocol for use of gene therapy in newborn infants. This chapter describes a strategy for the use of neonatal gene therapy in the treatment of inherited disorders and presents preclinical neonatal gene therapy data for two inherited disorders, metachromatic leukodystrophy (MLD) and hypophosphata‐ sia (HPP). We also discuss the utility, advantages, problems and the potential of neonatal gene

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 × 10<sup>11</sup> 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.

therapeutic approaches for the treatment of inherited disorders.

192 Selected Topics in Neonatal Care

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


**Table 1.** Advantages of neonatal gene therapy.

#### **4. Application of neonatal gene therapy**

#### **4.1. Neonatal gene therapy for metachromatic leukodystrophy**

Metachromatic leukodystrophy is an inherited, autosomal recessive lysosomal storage dis‐ ease (LSD) caused by a deficiency in the lysosomal enzyme arylsulfatase A (ASA), which catalyzes the degradation of galactosyl‐3‐sulfate ceramide (sulfatide (Sulf)), a major myelin sphingolipid [19]. This disease is characterized by myelin degeneration, mainly in the CNS, and clinically by progressive motor and mental deterioration that is ultimately lethal. Therefore, the major target organ for treatment of this disease is the CNS, and the aim is to arrest or reverse the progression of the neurological symptoms. A major obstacle, how‐ ever, is the BBB, which limits delivery of systemically administered therapeutic molecules to the brain [14]. It is therefore hoped that systemic administration of an AAV vector harbor‐ ing ASA during the neonatal period would be useful for treating the CNS. We previously showed that a single systemic injection of AAV vector encoding human ASA (AAV/hASA) into neonatal ASA knockout (MLD) mice results in the wide distribution of ASA in the brain and correction of the biochemical and neurological phenotypes [20]. **Figure 2A** shows that a single systemic injection of AAV/hASA enables transduction of the CNS in neonates but not

in adults. Efficient hASA expression was detected in the brain of AAV/hASA treated at the neonatal period of MLD mice. PCR analysis confirmed that AAV vector genome was observed only in neonatal‐treated MLD mice. Moreover, sustained expression of hASA in plasma was detected for at least 30 weeks after intravenous injection into neonatal MLD mice, while only transient increase in plasma hASA was obtained when injected into either adult MLD mice or wild‐type C57Bl/6 mice (**Figure 2B**). Vector injection into adult NOD‐SCID mice led to sustained secretion of hASA into the circulation, suggesting that immune responses to hASA are a major hurdle for successful gene therapy in immunocompetent adult MLD mice. It thus appears that the systemic injection of AAV vector during the neonatal period is a potentially

Neonatal Gene Therapy for Inherited Disorders http://dx.doi.org/10.5772/intechopen.69218 195

Hypophosphatasia is an inherited disease caused by a deficiency of tissue‐nonspecific alkaline phosphatase (TNALP) [21, 22]. The major symptom of human HPP is hypomineralization,

**Figure 3.** X‐ray images of the whole bodies of TNALP knockout mice. Radiographic images were obtained on lFX1000 film (Fujifilm Corp., Tokyo, Japan) using a setup for analysis of small animals. The energy level was 25 kV, and the exposure time was 90 s for 10‐day‐old untreated TNALP knockout (A), normal wild‐type (B) and AAV/TNALP‐D10‐

useful means of treating neurological disorders.

treated TNALP knockout mice (C).

**4.2. Neonatal gene therapy for hypophosphatasia**

**Figure 2.** hASA expression of MLD mice following neonatal systemic administration of AAV/hASA vectors. (A) Fifty‐two weeks after AAV/hASA injection, hASA concentration in the brain was determined by an indirect sandwich enzyme‐ linked immunosorbent assay (ELISA) (left panel). DNA from the brain was extracted and analyzed using PCR with hASA‐specific primers (right panel). (B) hASA expression in plasma of AAV/hASA‐injected mice. hASA concentration in plasma was determined by ELISA. Sustained expression was observed after neonatal injection of AAV/hASA.

in adults. Efficient hASA expression was detected in the brain of AAV/hASA treated at the neonatal period of MLD mice. PCR analysis confirmed that AAV vector genome was observed only in neonatal‐treated MLD mice. Moreover, sustained expression of hASA in plasma was detected for at least 30 weeks after intravenous injection into neonatal MLD mice, while only transient increase in plasma hASA was obtained when injected into either adult MLD mice or wild‐type C57Bl/6 mice (**Figure 2B**). Vector injection into adult NOD‐SCID mice led to sustained secretion of hASA into the circulation, suggesting that immune responses to hASA are a major hurdle for successful gene therapy in immunocompetent adult MLD mice. It thus appears that the systemic injection of AAV vector during the neonatal period is a potentially useful means of treating neurological disorders.

#### **4.2. Neonatal gene therapy for hypophosphatasia**

catalyzes the degradation of galactosyl‐3‐sulfate ceramide (sulfatide (Sulf)), a major myelin sphingolipid [19]. This disease is characterized by myelin degeneration, mainly in the CNS, and clinically by progressive motor and mental deterioration that is ultimately lethal. Therefore, the major target organ for treatment of this disease is the CNS, and the aim is to arrest or reverse the progression of the neurological symptoms. A major obstacle, how‐ ever, is the BBB, which limits delivery of systemically administered therapeutic molecules to the brain [14]. It is therefore hoped that systemic administration of an AAV vector harbor‐ ing ASA during the neonatal period would be useful for treating the CNS. We previously showed that a single systemic injection of AAV vector encoding human ASA (AAV/hASA) into neonatal ASA knockout (MLD) mice results in the wide distribution of ASA in the brain and correction of the biochemical and neurological phenotypes [20]. **Figure 2A** shows that a single systemic injection of AAV/hASA enables transduction of the CNS in neonates but not

194 Selected Topics in Neonatal Care

**Figure 2.** hASA expression of MLD mice following neonatal systemic administration of AAV/hASA vectors. (A) Fifty‐two weeks after AAV/hASA injection, hASA concentration in the brain was determined by an indirect sandwich enzyme‐ linked immunosorbent assay (ELISA) (left panel). DNA from the brain was extracted and analyzed using PCR with hASA‐specific primers (right panel). (B) hASA expression in plasma of AAV/hASA‐injected mice. hASA concentration in plasma was determined by ELISA. Sustained expression was observed after neonatal injection of AAV/hASA.

Hypophosphatasia is an inherited disease caused by a deficiency of tissue‐nonspecific alkaline phosphatase (TNALP) [21, 22]. The major symptom of human HPP is hypomineralization,

**Figure 3.** X‐ray images of the whole bodies of TNALP knockout mice. Radiographic images were obtained on lFX1000 film (Fujifilm Corp., Tokyo, Japan) using a setup for analysis of small animals. The energy level was 25 kV, and the exposure time was 90 s for 10‐day‐old untreated TNALP knockout (A), normal wild‐type (B) and AAV/TNALP‐D10‐ treated TNALP knockout mice (C).

rickets or osteomalacia, although the clinical severity is highly variable. Patients with infantile HPP may appear normal at birth but gradually develop rickets before reaching 6 months of age. Neonatal gene therapy is a promising strategy for treating infantile HPP by preventing early onset. We have shown that the phenotype of TNALP knockout mice [23–25], which mim‐ ics the severe infantile form of HPP, can be prevented by a single neonatal injection of AAV vector encoding bone‐targeted TNALP in which a deca‐aspartate tail is linked to the C‐termi‐ nus of soluble TNALP (AAV/TNALP‐D10). Sustained expression of TNALP and phenotypic correction of TNALP knockout mice were observed following the neonatal gene therapy [26]. X‐ray analysis showed that treated TNALP knockout mice grow as well as normal wild‐type mice (**Figure 3**).

and HPP model mice. We also thank Dr. Tae Matsumoto and Dr. Yukihiko Hirai for joint research and helpful discussions. This chapter was supported in part by grants from the Ministry of Health and Welfare of Japan and the Ministry of Education, Science and Culture

Neonatal Gene Therapy for Inherited Disorders http://dx.doi.org/10.5772/intechopen.69218 197

Department of Biochemistry and Molecular Biology, Division of Gene Therapy Research

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Center for Advanced Medical Technology, Nippon Medical School, Japan

of Japan.

**Author details**

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