Summary and Future Directions

Preclinical studies have demonstrated the feasibility of introducing an exogenous gene into the muscle of the developing fetus, although the optimal timing and best mode of delivery have yet to be defined. In utero gene therapy is still in its infancy for clinical application. While there are many potential advantages, the various safety and ethical implications must also be considered. With advanced techniques such as fetoscopy, ultrasound, and fetal surgical methods, it is possible to achieve in utero gene delivery while limiting the invasiveness of the procedure to both the fetus and the mother. While the aim of in utero gene therapy is to provide an optimal therapeutic benefit, every attempt must be made to minimize potential complications. Therefore, careful monitoring for birth defects and any long term side effects of in utero gene transfer will be required.

Important perinatal considerations of in utero gene transfer to the muscle include minimizing any increased risk of preterm labor, infection, and fetal loss. Human clinical approaches to fetal muscle have included fetal muscle biopsy (Heckel et al. 1999; Kuller et al. 1992) and ultrasound guided fetal gluteal muscle injection of corticosteroid, employed to improve fetal lung maturation (Ljubic et al. 1999). Therefore, the surgical approaches that would be required for human in utero gene transfer have precedence.

Some of the many factors concerning optimal timing of injecting the viral vector include prevention of insertional mutagenesis, avoidance of integration into the germ line and avoidance of deleterious immunity. Insertional mutagenesis, which could possibly lead to oncogenesis, can be avoided by using vectors that do not integrate into the host cell genome. Germ line integration can be minimized by injecting the vector into fetal tissue after the process of gonadal compartmentaliza-tion is completed in the fetus. In mouse, primordial germ cells complete colonization into gonadal primordium by E13 (Hogan et al. 1994). Similarly in humans the primordial germ cells are completely compartmentalized by the seventh week of gestation (GillmanJ 1948). By choosing the appropriate fetal developmental stage for gene delivery, germ line integration can be minimized. It is clear from the variable results to date that further studies of immunity and the development of tolerance in the setting of muscle gene transfer in utero are required.

One of the drawbacks of postnatal gene therapy is hepatic toxicity. Although some in utero studies in guinea pigs have shown liver transduction (Senoo et al. 2000), which could be due to the choice of the promoter, most evidence suggests low levels of liver transduction following in utero muscle or systemic gene delivery (Bilbao et al. 2005a, b; Boyle et al. 2001). Similar to AAV vector-mediated gene transduction in utero, AAV vector-mediated gene delivery in neonatal mice yielded significantly lower levels of liver transduction compared to gene delivery in adult mice (Wang et al. 2008, 2005). While additional studies are needed to understand the mechanisms of low liver transduction in most in utero studies, the high prolifera-tive state and unique properties of the fetal liver during development could play a role (Lansdorp et al. 1993; O'Donoghue and Fisk 2004; Taylor et al. 2002). Another consideration that impacts the ultimate safety of in utero gene transfer is the trans-placental spread of vector from the fetus to tissues of the pregnant female. Studies have shown the presence of antiadenovirus and antitransgene antibodies in maternal serum (Bilbao et al. 2005b). Although these antibodies were not neutralizing (Bilbao et al. 2005b), immune tolerance, liver toxicity and other consequences need to be considered.

In this chapter, we have given the results of the studies on the unique potential, the different vectors used to date in experimental studies, and the challenges of in utero muscle gene transfer. Future advances will depend on continued vector development, a better understanding of effects on target and nontarget tissues and the induction of immunity and strategies to safely enhance efficient gene transduction of the muscle tissue for long-lasting therapeutic benefit.

Acknowledgment PRC is supported by VA resources (Pittsburgh VA Healthcare System, Pittsburgh, PA).

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