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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. RNA Interference Induced Gene Silencing
  5. Using RNAi to Disrupt Fertility
  6. Novel RNAi Delivery Vehicle
  7. Modification of the AAV Capsid to Target the Brain
  8. Improved Design of siRNAs to Enhance Specificity
  9. Concluding Remarks
  10. Conflicts of interest
  11. Funding Sources
  12. References

Population control of feral animals is often difficult, as it can be dangerous for the animals, labour intensive and expensive. Therefore, a useful tool for control of animal populations would be a non-surgical method to induce sterility. Our laboratories utilize methods aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A useful framework for design of a new approach will be the combination of these methods with the intended goal to produce a technique that can be used to non-invasively sterilize cats and dogs. For this approach to succeed, it has to meet several conditions: the target gene must be essential for fertility; the method must include a mechanism to effectively and specifically silence the gene of interest; the method of delivering the silencing agent must be minimally invasive, and finally, the silencing effect must be sustained for the lifespan of the target species, so that expansion of the population can be effectively prevented. In this article, we discuss our work to develop gene silencing technology to induce sterility; we will use examples of our previous studies demonstrating that this approach is viable. These studies include (i) the use of viral vectors able to disrupt reproductive cyclicity when delivered to the regions of the brain involved in the control of reproduction and (ii) experiments with viral vectors that are able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. RNA Interference Induced Gene Silencing
  5. Using RNAi to Disrupt Fertility
  6. Novel RNAi Delivery Vehicle
  7. Modification of the AAV Capsid to Target the Brain
  8. Improved Design of siRNAs to Enhance Specificity
  9. Concluding Remarks
  10. Conflicts of interest
  11. Funding Sources
  12. References

An inexpensive non-invasive 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 controlling reproduction 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 (Funes et al. 2003; de Roux et al. 2003; Semple et al. 2005; d'Anglemont de Tassigny et al. 2007; Clarkson et al. 2008; Topaloglu et al. 2008, 2012; Silveira et al. 2010). 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 remain active indefinitely, permanent sterility would be the outcome of a single systemic administration.

RNA Interference Induced Gene Silencing

  1. Top of page
  2. Contents
  3. Introduction
  4. RNA Interference Induced Gene Silencing
  5. Using RNAi to Disrupt Fertility
  6. Novel RNAi Delivery Vehicle
  7. Modification of the AAV Capsid to Target the Brain
  8. Improved Design of siRNAs to Enhance Specificity
  9. Concluding Remarks
  10. Conflicts of interest
  11. Funding Sources
  12. References

The cellular mechanism known as RNAi results in post-transcriptional gene regulation in both plants and animals; the most basic unit of the RNAi system is the microRNA (miRNA) (Krol et al. 2010). To harness the RNAi system, artificial miRNAs known as small interfering RNAs (siRNAs) have been developed (Provost et al. 2002; Cullen 2005). Natural miRNAs and siRNAs are similarly short double-stranded RNAs (~22 nt) (Cullen 2005). The miRNA is synthesized as part of a structure known as a primary miRNA containing stem-loop regions. The primary miRNA is processed by the enzyme Drosha, an RNase III, releasing a stem-loop pre-miRNA structure (Lee et al. 2003). A RNA molecule known as the short hairpin RNA (shRNA) is the artificial version of the stem-loop pre-miRNA. A second RNase III enzyme known as Dicer acts on both the pre-miRNA and the shRNA to release the natural miRNA or the artificial siRNA (Lee et al. 2003; Gregory et al. 2004). The natural miRNA and the artificial siRNA enter the cellular machinery in an identical manner. The antisense or guide strand of the miRNA is complementary to the mRNA and is incorporated into a ribonucleoprotein RNA-induced silencing complex (RISC) (Khvorova et al. 2003; Schwarz et al. 2003; Cullen 2005). The degree of complementarity of the miRNA or siRNA to the target mRNA determines the subsequent processing pathway. High complementarity results in the cleavage of the mRNA and suppression of transcription, and low complementarity and imperfect binding leads to translational repression and mRNA destabilization (Guo et al. 2010; Boudreau et al. 2011). This type of interaction often occurs in the 3′-untranslated region (UTR); it is thought that pairing with as few as 6–7 nucleotides of the miRNA is sufficient to achieve silencing (Lewis et al. 2005; Boudreau et al. 2011). As the scientific community learns more about how miRNAs are naturally produced and how they achieve gene silencing, miRNA-mediated RNAi is becoming a more useful tool to silence virtually any gene for therapeutic purposes.

Using RNAi to Disrupt Fertility

  1. Top of page
  2. Contents
  3. Introduction
  4. RNA Interference Induced Gene Silencing
  5. Using RNAi to Disrupt Fertility
  6. Novel RNAi Delivery Vehicle
  7. Modification of the AAV Capsid to Target the Brain
  8. Improved Design of siRNAs to Enhance Specificity
  9. Concluding Remarks
  10. Conflicts of interest
  11. Funding Sources
  12. References

Suppression of a gene known as Enhanced at Puberty 1 (EAP1) using RNAi demonstrated that EAP1 is required for normal cyclicity in both rats and non-human primates, and simultaneously provided evidence that RNAi can be exploited to disrupt fertility. In female rats a region-specific decrease in EAP1 expression, induced by RNAi, delayed puberty and disrupted adult reproductive cyclicity (Heger et al. 2007). The shRNA complementary to Eap1 mRNA was inserted in the 3′-long terminal repeat of a lentiviral (LV) vector. The shRNA-LV vector was administered to juvenile 23-day-old female rats by bilaterally microinjections into the preoptic area (POA) (Heger et al. 2007). In rats, most of the GnRH neurons involved in the hypothalamic control of gonadotropin secretion by the pituitary are located in the POA-anteroventral periventricular nucleus (AVPV) and an intact AVPV is required for the recurrent surges of gonadotropin released during the rat oestrous cycle (Simerly 2002; Herbison 2006). LV particles devoid of inhibitory shRNAs and containing an enhanced green fluorescent protein (eGFP)-reporter gene were injected into control animals (Heger et al. 2007). Cells located along the lateral borders of the AVPV were transduced and exhibited expression of eGFP. The morphological appearance of infected cells was that of neurons, and some of the infected cells were identified by immunohistochemistry as GnRH neurons. The shRNA was shown to be effective in suppressing Eap1 expression by the reduced content of EAP1 immunoreactive material observed in infected cells vs. non-infected cells.

In rats in which expression of the Eap1 gene was knocked-down by shRNA in the POA, the time of puberty (defined by the time of first ovulation) was delayed as compared to LV eGFP-injected rats (Heger et al. 2007). Rats also exhibited a disrupted oestrous cycle following Eap1 gene knock-down including prolonged episodes in oestrus, and reduced plasma LH, FSH and estradiol levels.

Using non-human primates, a species closer to humans, EAP1 was also shown to be required for reproductive cyclicity. Improvements in RNAi technology allowed the production of an artificial primary miRNA, which permitted a more natural processing of the inhibitory RNA expressed from the LV construct. Because the LV construct employed contains the reporter gene eGFP, the cells in the monkey hypothalamus that had been transduced were identified by eGFP immunohistofluorescence (Dissen et al. 2012a). Throughout the medial basal hypothalamus-arcuate nucleus region cells positive for eGFP were found in animals receiving correctly placed injections. There were no signs of an inflammatory reaction in the transduced area, as the distribution of cell populations identified by Hoechst staining of cell nuclei was normal and astrogliosis was absent (Dissen et al. 2012a). Similar to the observation in the rat, cells infected with LV particles carrying RNAi had a lower content of EAP1 immunoreactive material than non-infected cells.

Monkeys of reproductive age cycling normally were used for this study. Animals that received the control miRNA LV particles continued to cycle following the injections. Menstrual cyclicity was completely arrested in monkeys that received LV particles containing the EAP1 miRNA, within the target area (Dissen et al. 2012a). The importance of transducing the correct region of the brain was demonstrated by the finding that animals in which the microinjections were misplaced continued to cycle after the injection.

The studies described earlier clearly demonstrate that RNAi is capable of disrupting female reproductive cyclicity. However, the LV vector delivering RNAi had to be directly targeted to the intracerebral location of interest to ensure effectiveness. This delivery method is not only undesirable, but also impractical when the goal is to induce infertility in dogs and cats. Because one of our groups succeeded in targeting genes to the brain vasculature using a novel gene delivery method, our laboratories have joined efforts to devise an approach that can be effectively used to overcome this major limitation of gene delivery.

Novel RNAi Delivery Vehicle

  1. Top of page
  2. Contents
  3. Introduction
  4. RNA Interference Induced Gene Silencing
  5. Using RNAi to Disrupt Fertility
  6. Novel RNAi Delivery Vehicle
  7. Modification of the AAV Capsid to Target the Brain
  8. Improved Design of siRNAs to Enhance Specificity
  9. Concluding Remarks
  10. Conflicts of interest
  11. Funding Sources
  12. References

The aforementioned studies might suggest that EAP1 would be a good target for RNAi-mediated silencing of reproduction; however, EAP1 expression is widespread throughout the body (Su et al. 2004) (database accessed via http://en.wikipedia.org), thereby limiting the specificity of a systemic approach to gene silencing. The target gene for RNAi-mediated suppression of fertility should be essential for fertility, exhibit tissue-specific expression and not be required for processes other than reproduction. A gene that meets these criteria is the Kiss1 gene (Funes et al. 2003; de Roux et al. 2003; Semple et al. 2005; Clarkson et al. 2008; Topaloglu et al. 2012). Kiss1 has a limited tissue distribution, including the placenta (Su et al. 2004) (database accessed via http://en.wikipedia.org), the hypothalamus (Gottsch et al. 2004; Clarkson et al. 2009) and the ovary (Castellano et al. 2006; Gaytan et al. 2009). Confirmation that Kiss1 is an ideal target candidate comes from studies of mice lacking Kiss1, which are infertile and yet exhibit no other gross abnormalities (d'Anglemont de Tassigny et al. 2007).

For permanent RNAi-based, viral-mediated gene therapy incorporation of the viral genome into the host genome, as is the case of LV vectors, is advantageous. However, there is also a risk of genomic alterations that could lead to cancer or other gene mutations. A vector that does not incorporate into the host genome, such as the recombinant adeno-associated virus (AAV) vector would be a better choice for an RNAi-mediated sterilant. Recombinant AAV differs from its wild-type counterparts in that the viral genome takes up residence in the cell nucleus as a double-stranded episome, which does not insert into the host genome. In spite of existing as an episome, the AAV genome can drive transgene expression for long periods of time. Transgenes from AAV vectors can be detected in many different tissues of rodents throughout the lifespan of the animal (Kaplitt et al. 1994; McCown et al. 1996; Xiao et al. 1996); in non-human primates, they have been detected for as long as 10 years (Dissen et al. 2012b). Another factor that has contributed to the acceptance of the AAV vector for gene therapy is that the vector is derived from a non-pathogenic virus (Mueller and Flotte 2008; Mingozzi and High 2011).

An important feature of the recombinant AAV is that it can be administered in a non-invasive manner such as intravenous injections. Systemic administration of AAV is minimally invasive, but there are no known serotypes that target the brain selectively. Serotypes are defined as isolates of AAV that do not exhibit cross-reaction with neutralizing sera from any other known serotype (Wu et al. 2006). Importantly, AAV serotypes have been found to exhibit unique tropism for different tissues throughout the body. The various tropisms are achieved by the use of different cell surface receptors to gain entry into the host cell (Wu et al. 2006). The most extensively studied serotype is AAV2; hundreds of publications have detailed both pre-clinical and clinical uses of AAV2 to deliver transgenes to the liver (the primary target for AAV2) and various other tissues throughout the body (Mueller and Flotte 2008). Unfortunately, very little of the virus crosses the blood brain barrier and enters the brain. The AAV2 vector is targeted to specific cell types, via a heparin sulphate proteoglycan (HSPG) binding domain on the surface of the AAV capsid protein (Bartlett et al. 2000).

Modification of the AAV Capsid to Target the Brain

  1. Top of page
  2. Contents
  3. Introduction
  4. RNA Interference Induced Gene Silencing
  5. Using RNAi to Disrupt Fertility
  6. Novel RNAi Delivery Vehicle
  7. Modification of the AAV Capsid to Target the Brain
  8. Improved Design of siRNAs to Enhance Specificity
  9. Concluding Remarks
  10. Conflicts of interest
  11. Funding Sources
  12. References

The tropism of AAV2 can be redirected by targeting the HSPG binding domain of the capsid protein. This modification has been achieved by inserting a peptide sequence at the arginine 588 of the capsid protein (Muller et al. 2003). The new tropism of the virus is dependent on the peptide sequence inserted. A technique termed biopanning has shown great promise for selecting such peptide sequences (Work et al. 2002). The essence of biopanning is that a random peptide library is displayed on the binding domain of a bacteriophage; this phage-display library is injected into the vasculature of the target species, then the tissues of interest are collected and the adherent phage is extracted (Work et al. 2006; Chen et al. 2009). The biopanning technique was used by one of our groups to select an epitope that directs the binding of AAV2 to the vasculature of the brain (Chen et al. 2009). The virus carrying this epitope showed targeting of the brain several orders of magnitude greater than the wild-type virus, and a viral reporter gene was detected in cells positive for the neuronal marker NeuN (Chen et al. 2009). The colocalization of the reporter gene, with the NeuN marker suggests that the virus not only targeted the brain vasculature but also entered and transduced the underlying neuropil.

Generally, the insertion of a peptide at arginine 588 alters the capsid receptor binding characteristics without compromising virus viability (Muller et al. 2003; Perabo et al. 2006; Work et al. 2006). In previous studies, the entire brain was used for the biopanning experiment resulting in several candidate peptides that were used for targeting the virus to the brain (Chen et al. 2009). In current studies, our groups used biopanning to identify a peptide epitope able to redirect the tropism of AAV2 to the medial basal hypothalamus, the region where Kiss1 expressing neurons, critical for reproduction, are located. This region of the brain is more accessible to systemically delivered molecules via the vasculature because it does not appear to be isolated by a fully functional blood brain barrier, a feature that allows significant transfer of macromolecules from the bloodstream to the hypothalamic parenchyma (Broadwell and Brightman 1976; Herde et al. 2011).

Improved Design of siRNAs to Enhance Specificity

  1. Top of page
  2. Contents
  3. Introduction
  4. RNA Interference Induced Gene Silencing
  5. Using RNAi to Disrupt Fertility
  6. Novel RNAi Delivery Vehicle
  7. Modification of the AAV Capsid to Target the Brain
  8. Improved Design of siRNAs to Enhance Specificity
  9. Concluding Remarks
  10. Conflicts of interest
  11. Funding Sources
  12. References

The ability of siRNAs to specifically suppress expression of target mRNA determines the effectiveness of RNAi as a gene silencing tool. A negative feature of siRNAs is the suppression of unintended mRNAs, an effect known as off-target silencing (Chi et al. 2003; Jackson et al. 2003; Semizarov et al. 2003). This type of silencing likely occurs when the region of the siRNA known as the ‘seed’ (nucleotides 2–8 of the antisense strand) specifically pairs with the 3′-UTR sequences of mRNAs other than the intended target and results in destabilization or repression of those transcripts (Birmingham et al. 2006; Anderson et al. 2008). Off-target silencing has even been associated with, and thought to result in, toxic phenotypes (Fedorov et al. 2006). The magnitude of siRNA off-targeting has been shown to be directly related to the frequency of hexamer seed complements present in the 3′-UTR transcriptome (Boudreau et al. 2011). As a result of these observations, the siRNA design scheme has been developed to prioritize seed specificity (known as safe-seeds) as a means to improve the safety profile of therapeutic RNAi sequences. The safe-seeds method of sequence selection proved successful in identifying siRNAs that silenced target gene expression, induced minimal seed-related off-targeting and were well-tolerated in mice (Boudreau et al. 2011). We have now used the safe-seeds method to design siRNAs against Kiss1. In ongoing studies, we are determining whether one of these siRNAs can be delivered by a peptide-modified AAV2 vector to the hypothalamus and interfere with fertility in rats.

Concluding Remarks

  1. Top of page
  2. Contents
  3. Introduction
  4. RNA Interference Induced Gene Silencing
  5. Using RNAi to Disrupt Fertility
  6. Novel RNAi Delivery Vehicle
  7. Modification of the AAV Capsid to Target the Brain
  8. Improved Design of siRNAs to Enhance Specificity
  9. Concluding Remarks
  10. Conflicts of interest
  11. Funding Sources
  12. References

Proof-of-principle that RNAi can be used to cause sterility has been provided by studies in which RNAi targeted against a gene involved in the control of reproduction was delivered to the hypothalamus via intracerebral administration of the miRNA encoding LV vector. This procedure disrupted reproductive cyclicity in both female rats and non-human primates. Because in non-human primates ovulation was blocked, silencing of this single gene appears to cause a state of infertility. Current efforts are aimed at devising a modified AAV2 vector to selectively, or even specifically, target the hypothalamus upon systemic administration. We envision that this method will provide the means to silence genes essential for reproduction in a non-invasive, effective and sustained manner in dogs and cats. Figure 1 is a schematic representation/summary of this experimental plan. We also anticipate that these studies will provide the basis for new delivery strategies to the brain for basic research purposes and emerging therapies.

image

Figure 1. Schematic diagram of the research plan summarized in the article. The goal is to use a peptide-modified adeno-associated virus (AAV) vector to suppress fertility in cats and dogs. A bacteriophage library is administered by intravenous systemic injection (1) to adult females. The bacteriophage that homes to the hypothalamus (2) is isolated, purified and administered to naive animals (3). This procedure (termed bio-panning) is repeated until convergence of a single peptide sequence is observed. The DNA coding for the peptide sequence is inserted (4) into the DNA sequence encoding the capsid protein for the AAV vector. An siRNA/miRNA sequence that is the most effective at suppressing expression of the target mRNA in vitro is selected (5), and inserted into the genome of the AAV vector (6). The peptide-modified siRNA/miRNA AAV vector is then administered intravenously (7) to the target species; either cats or dogs. While untreated animals will continue to produce offspring normally, GnRH is reduced in the treated animals resulting in infertility

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Funding Sources

  1. Top of page
  2. Contents
  3. Introduction
  4. RNA Interference Induced Gene Silencing
  5. Using RNAi to Disrupt Fertility
  6. Novel RNAi Delivery Vehicle
  7. Modification of the AAV Capsid to Target the Brain
  8. Improved Design of siRNAs to Enhance Specificity
  9. Concluding Remarks
  10. Conflicts of interest
  11. Funding Sources
  12. References

This work was supported by the Michelson Foundation and the Division of Program Coordination, Planning, and Strategic Initiatives (DPCPSI) and the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health through Grant Number OD011092.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. RNA Interference Induced Gene Silencing
  5. Using RNAi to Disrupt Fertility
  6. Novel RNAi Delivery Vehicle
  7. Modification of the AAV Capsid to Target the Brain
  8. Improved Design of siRNAs to Enhance Specificity
  9. Concluding Remarks
  10. Conflicts of interest
  11. Funding Sources
  12. References
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