**2. AAV vectors for CNS disorders**

#### **2.1. Advantages of AAV vectors for gene transfer to CNS**

A variety of both viral and non-viral vectors have been applied to the effort to transfer genes into the CNS. Among these gene transfer methods, AAV vectors have emerged as a particularly promising tool for gene delivery. There are many advantages to using AAV vectors for transduction of the CNS [3]. First, AAV is itself not pathogenic and has received a P1 and P1A recombinant DNA safety classification. It is therefore easy to use AAV vectors without specific facilities. Second, because AAV vectors have the ability to transduce non-dividing cells, they are a suitable means for delivering transgenes to non-dividing neuronal cells [4]. Third, longterm expression of transgenes with little immunogenicity or toxicity can be achieved using AAV vectors. It was reported that there was a gradual decline in the number of transduced cells when the cytomegalovirus (CMV) immediate-early promoter was used [5]. However, with the use of cellular or hybrid promoters, such as the chicken beta-actin/CMV promoter, transgene expression was sustained. Indeed, we found that following administration to mice of an AAV vector in which the CAG promoter drove the green fluorescent protein (GFP) gene, GFP expression was sustained for more than one and a half years, or nearly the entire life span of the mice [6]. In addition, we are now using several AAV vector serotypes (mainly from AAV serotype 1 to 12), depending on the target. On the other hand, a disadvantage of AAV vectors is the size limitation of the transgene. Since AAVs package a ∼4.7-kb genome, it is better to have 4.7 kb between the inverted terminal repeats at the 5' and 3' ends [7].

#### **2.2. How to cross the BBB?**

Adeno-associated virus (AAV) is a member of the family Parvoviridae that has been widely used as a vector for gene delivery. AAV is a small, non-enveloped single-stranded DNA virus with a genome of approximately 4.7 kb [1]. The AAV genome consists of three open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs). It is a dependovirus because it requires helper functions from other viruses such as adenovirus or herpes simplex virus for its replication. AAV is a suitable gene transfer tool because of its safety due to a lack of pathogenicity, its ability to transduce both dividing and non-dividing cells, and its minor immune response. Among the more than 100 nonredundant AAV genotypes that have been identified, 12 AAV serotypes with unique properties have been used to produce most

AAV vectors are powerful tools that are able to mediate gene transfer to the CNS, thanks to their safety and ability to transduce non-dividing neuronal cells. Consequently, they hold great potential for use in therapeutic gene delivery strategies for the treatment of neurological disorders. Although a breakthrough treatment has remained elusive, current approaches are now considerably safer and potentially much more effective than in the past. In this chapter, we discuss how to administer vectors across the BBB, focusing especially on potential routes for administration of AAV vectors and promising strategies for application of AAV vectors in

A variety of both viral and non-viral vectors have been applied to the effort to transfer genes into the CNS. Among these gene transfer methods, AAV vectors have emerged as a particularly promising tool for gene delivery. There are many advantages to using AAV vectors for transduction of the CNS [3]. First, AAV is itself not pathogenic and has received a P1 and P1A recombinant DNA safety classification. It is therefore easy to use AAV vectors without specific facilities. Second, because AAV vectors have the ability to transduce non-dividing cells, they are a suitable means for delivering transgenes to non-dividing neuronal cells [4]. Third, longterm expression of transgenes with little immunogenicity or toxicity can be achieved using AAV vectors. It was reported that there was a gradual decline in the number of transduced cells when the cytomegalovirus (CMV) immediate-early promoter was used [5]. However, with the use of cellular or hybrid promoters, such as the chicken beta-actin/CMV promoter, transgene expression was sustained. Indeed, we found that following administration to mice of an AAV vector in which the CAG promoter drove the green fluorescent protein (GFP) gene, GFP expression was sustained for more than one and a half years, or nearly the entire life span of the mice [6]. In addition, we are now using several AAV vector serotypes (mainly from AAV serotype 1 to 12), depending on the target. On the other hand, a disadvantage of AAV vectors is the size limitation of the transgene. Since AAVs package a ∼4.7-kb genome, it is better to

expression vectors [2].

106 Gene Therapy - Principles and Challenges

CNS disorders.

**2. AAV vectors for CNS disorders**

**2.1. Advantages of AAV vectors for gene transfer to CNS**

have 4.7 kb between the inverted terminal repeats at the 5' and 3' ends [7].

To apply gene therapy to the treatment of CNS disorders, there are two immediate problems that must be solved: One is how to cross the BBB, which is a physical and biochemical barrier that precisely regulates the ability of endogenous and exogenous substances to accumulate within brain tissue [8, 9], and the second is how to distribute the transgene to the entire brain. In some cases, we have to treat the whole brain, including the peripheral nervous system (PNS). To transfer genes across the BBB, there are two main approaches: brain-directed local therapy and less invasive systemic therapy. Table 1 shows possible approaches of gene delivery to CNS using AAV vectors.


**Table 1.** Possible approaches of gene delivery to CNS using AAV vectors
