Applying gene silencing technology to contraception.

An inexpensive noninvasive method to permanently sterilize dog and cats would be of great benefit to both humans and animals. In the case of feral populations of animals such a sterilant would provide a means of population control without euthanizing the adult animals. By reducing the population of feral animals, there would be less stress on the environment and the animals would hopefully suffer less from malnutrition and disease. Further, the chances of serving as a vector for human disease would also be reduced. In the case of pet populations, an inexpensive sterilant would hopefully result in increased pet sterilization and a corresponding decrease in the number of unwanted pets, strays and abandoned dogs and cats which are often euthanized. Reproductive success, in all mammals requires the pulsatile release of GnRH from the hypothalamus. Thus the control of GnRH release is a natural control point that can be exploited for the purpose of producing a sterilant. The seminal event underlying the initiation and progression of puberty is the increase in the pulsatile release of GnRH (Ojeda et al., 2010); once firmly established, GnRH pulsatility is central to the normalcy of reproductive cyclicity in females and fertility in both sexes. The GnRH neuronal network is mostly located in the medial basal hypothalamus in humans, monkeys, sheep, dogs and cats (Barry and Dubois, 1975;Belda et al., 2000;Heger et al., 2007;Dissen et al., 2012a). Neuronal (Kordon et al., 1994;Ojeda and Terasawa, 2002) and glial ( Ojeda and Terasawa, 2002;Ojeda et al., 2003) inputs to GnRH neurons provide coordination to the network. Neuronal inputs act trans-synaptically to regulate GnRH secretion using either excitatory or inhibitory neurotransmitters/neuromodulators ( Terasawa and Fernandez, 2001;Ojeda and Terasawa, 2002;Plant and Witchel, 2006), while the glial component is predominantly facilitatory, and is to a significant extent represented by growth factors that directly or indirectly stimulate GnRH secretion (Ojeda et al., 2003;Ojeda and Skinner, 2006;Lomniczi and Ojeda, 2009). The episodic release of GnRH is a mode of hormonal output that is essential for reproduction, and is driven by a subset of neurons located in the arcuate nucleus of the medial basal hypothalamus. These cells produce Kisspeptin, Neurokinin B (NKB), and Dynorphin (Wakabayashi et al., 2010;Navarro et al., 2011); because of this, they are known as KNDy neurons (Lehman et al., 2010;Navarro et al., 2011). The release of NKB from KNDy neurons stimulates kisspeptin release from the same type of neurons by a specific receptor mediated activation (Wakabayashi et al., 2010;Navarro et al., 2011). Key to this system is a phase-delayed inhibitory feedback of dynorphin on NKB release to produce an oscillatory release of NKB and kisspeptin (Wakabayashi et al., 2010;Navarro et al., 2011). Deletion of the gene or receptor for kisspeptin or NKB has shown that both are required for normal fertility (de Roux et al., 2003;Funes et al., 2003;Semple et al., 2005;d’Anglemont, X et al., 2007;Clarkson et al., 2008;Topaloglu et al., 2008;Silveira et al., 2010;Topaloglu et al., 2012). The absolute requirement of kisspeptin and NKB for fertility makes them targets for knockdown by RNA interference (RNAi) that would result in infertility and potentially sterility. Our goal in this study is to develop a permanent method of sterility, by combining gene silencing with a gene therapy. The gene silencing technique will specifically target the gene of interest; while the gene therapy delivery system will target the specific brain region that is critical for fertility. Because the gene therapy vector is expected to remains active indefinitely, permanent sterility would be the outcome of a single systemic administration.