Abstract
Recent articles have appeared in the literature reporting use of adeno-associated virus vectors (AAV) expressing phenylalanine hydroxylase in animal trials and suggesting its use in treatment of phenylketonuria (PKU) as a form of gene therapy However, agents used in gene therapy to deliver genes are not site-specific and DNA is may be put in the wrong place, causing damage to the organism. The adverse immunogenicity of AAVs also needs to be reconsidered. This letter is written to discuss present unreadiness for Phase 1 clinical trials of gene therapy of PKU. Turk Jem 2009; 13: 18-9
Key words: Gene therapy, phenylketonuria, adeno-associated vector, phase 1 clinical trial

Özet
Son zamanlarda, bazı yayınlarda adeno-ilişkili virüs ve vektörlerle (AAV) Fenilalanin Hidroksilaz geninin Fenilketonüri hastalarına klinik aktarımının yararlı olabileceğini düşündüren hayvan denemeleri yayınlanmıştır.Bu makale DNA'da bölgeye-özgün integrasyon ve immün yan etkiler kavramlarını örnekleyerek, planlanacak Faz 1 klinik çalışmalar öncesi yapılması gereken diğer bazı gerekli diğer araştırmaları hatırlatmaktadır. Turk Jem 2009; 13: 18-9
Anahtar kelimeler: Gen tedavisi, fenilketonüri, adeno-ilişkili vektör, faz 1 klinik çalışma

Abstract
Recent articles have appeared in the literature reporting use of adeno-associated virus vectors (AAV) expressing phenylalanine hydroxylase in animal trials and suggesting its use in treatment of phenylketonuria (PKU) as a form of gene therapy However, agents used in gene therapy to deliver genes are not site-specific and DNA is may be put in the wrong place, causing damage to the organism. The adverse immunogenicity of AAVs also needs to be reconsidered. This letter is written to discuss present unreadiness for Phase 1 clinical trials of gene therapy of PKU. Turk Jem 2009; 13: 18-9
Key words: Gene therapy, phenylketonuria, adeno-associated vector, phase 1 clinical trial

Özet
Son zamanlarda, bazı yayınlarda adeno-ilişkili virüs ve vektörlerle (AAV) Fenilalanin Hidroksilaz geninin Fenilketonüri hastalarına klinik aktarımının yararlı olabileceğini düşündüren hayvan denemeleri yayınlanmıştır.Bu makale DNA'da bölgeye-özgün integrasyon ve immün yan etkiler kavramlarını örnekleyerek, planlanacak Faz 1 klinik çalışmalar öncesi yapılması gereken diğer bazı gerekli diğer araştırmaları hatırlatmaktadır. Turk Jem 2009; 13: 18-9
Anahtar kelimeler: Gen tedavisi, fenilketonüri, adeno-ilişkili vektör, faz 1 klinik çalışma

Articles have recently appeared in the literature reporting the use of adeno-associated virus vectors (AAV) expressing phenylalanine hydroxylase (PAH) in animal trials and suggesting its use in treatment of phenylketonuria (PKU) as a form of gene therapy (1-3). However, agents used in gene therapy to deliver genes are not site-specific, and DNA is may be put in the wrong place, causing damage to the organism. Such effects have recently been observed with the phiBT1 phage during attempts to integrate the PAH gene into the human genome. It was observed that the Streptomyces phage phiBT1 integrase catalyzes recombination between phage attachment (att) attP and bacterial attB sites, resulting in phage DNA integration into the bacterial host genome in a unidirectional manner whereas, in the presence of multiple pseudo-attB and -attP sites in mammalian genomes, the recombination with wild-type attP and attB sequences occur on multiple sites. In fact, there were three pseudo-attP sites and two pseudo-attB sites in human cells, which are located in intergenic regions of five different chromosomes and are likely to end in non-site-specific integration (and thus mutation) (4). Therefor, predicting preferential DNA vector insertion sites is very important to understand the implications for functional genomics and gene therapy for AAV vector systems as well (5-7). Knowing the variables that affect integration for various vectors as well as AAV subtypes will theoretically allow researchers to choose the vector with the most utility for their specific purposes.
The three principal benefits from elucidating factors that affect preferences in integration are as follows:
• in gene therapy, it allows assessment of overall risks for activating an oncogene or inactivating a tumor suppressor gene that could lead to severe adverse effects years after treatment;
•  in genomic studies, it allows one to discern random from selected integration events;
• in gene therapy (as well as functional genomics), it facilitates design of vectors that are better targeted to specific sequences, which would be a significant advance in the art of transgenesis.
Thus it is advisable for the authors of animal studies attempting gene therapy of PKU by AAV that they FISH probe their AAV mediated gene integration sites most importantly on the human genome using human cell lines, and go even further to sequence these sites to elucidate any sequential structural characteristics that will lead to specific or nonspecific integration. Otherwise, unforeseen toxicity may lead to irreversible consequences (8).
It should also be noted that there is considerable evidence about the adverse immunogenicity of AAVs. The mechanisms and means to cope with this immune side effect also need further investigation before any human pharmacological attempts.

Address for Correspondence: Ayşegül Akbay Yarpuzlu, MD, Ankara University, Faculty of Medicine, Public Health, Ankara, Turkey E-mail: yarpuzlu@medicine.ankara.edu.tr Recevied: 23.03.2009 Accepted: 17.05.2009

References

1. Ding Z, Harding C, Rebuffat A, Elzaouk L, Wolff JA, Thöny B. Correction of murine PKU following AAV-mediated intramuscular expression of a complete phenylalanine hydroxylating system. Mol Ther 2008; 16: 673-81.
2. Jung SC, Park JW, Oh HJ, Choi JO, Seo Kİ, Park ES, Park HY. Protective effect of recombinant adeno-associated virus 2/8-mediated gene therapy from the maternal hyperphenylalaninemia in offsprings of a mouse model of phenylketonuria. J Korean Med Sci 2008; 23: 877-83.
3. Mochizuki S, Mizukami H, Ogura T, Kure S, Ichinohe A, Kojima K, Matsubara Y, Kobayahi E, Okada T, Hoshika A, Ozawa K, Kume A. Long-term correction of hyperphenylalaninemia by AAV-mediated gene transfer leads to behavioral recovery in phenylalanine mice. Gene Ther 2004: 11; 1081-6.
4. Chen L, Woo SL. Site-specific transgene integration in the human genome catalyzed by phiBT1 phage integrase. Hum Gene Ther 2008; 19: 143-51.
5. Hackett CS, Geurts AM, Hackett PB. Predicting preferential DNA vector insertion sites: implications for functional genomics and gene therapy. Genome Biol 2007; 8(Suppl 1): S12.
6. Campos SK, Barry MA. Current advances and future challenges in adenoviral vector biology and targeting. Curr Gene Ther 2007; 7: 189-204.
7. Kwon I, Schaffer DV. Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer. Pharm Res 2008; 25: 489-99.
8. Howard DB, Powers K, Wang Y, Harvey BK. Tropism and toxicity of adeno-associated viral vector serotypes 1,2,5,6,7,8,9 in rat neurons and glia in vitro. Virology. Author manuscript; available in PMC 2009 March 1.