Michelson Grants Research Findings

1. First demonstration of GnIH function in male and female feline gonads – inhibition of steroid production.
2. Successfully constructed an AAV-GnIH vector and demonstrated in vitro activity (increased RFRP expression).
3. AAV-GnIH single-dose administration to male rats conferred a significant, transient increase in body weight over the first 2 months post-administration.
4. AAV-GnIH single-dose administration to male rats failed to confer a significant biological effect on plasma T levels or on gonad size over the 3-month study period.

1. Generated siRNA against rat Kiss1 and Tac2 genes that encode for kisspeptin and neurokinin B, respectively.
2. Identified homing peptides to the rat hypothalamus using phage panning.
3. Incorporated siRNAs and homing peptides into an AAV2 vector and administered the construct to female rats IV; treated rats showed 20% reduction in Kiss1 RNA levels, disruption of estrous cycles, and delayed age at first ovulation, but not complete suppression of reproduction.
4. Cloned & verified DNA sequencing of Kiss1 gene in dogs and cats and determined that there are 4 regions of at least 17 nucleotides showing homology in both species that could be targets for RNA inhibition in both species.
5. Generated siRNAs that target the common coding regions of the cat and dog Kiss1 and Tac2 genes.
6. Demonstrated 2 large deletions in dog Kiss1 that are present in other canids but not in raccoons, mink, skunks or felids.

Project in progress

1. Identified and cloned canine and feline PIWIL1 and PIWIL2 and expressed as recombinant proteins in mammalian cells.
2. Identified differential patterns of PIWIL1 and PIWIL2 expression in immature (lower) vs. mature (higher) male dogs and cats.
3. Confirmed and expanded basic knowledge of piRNA/PIWI pathway involved in male gametogenesis in dogs and cats.
4. Developed new, improved methods and protocols to allow for longer in vitro cultivation of dog and cat male gonadal explants.

1. Investigators found that 30% of vaccinated and unvaccinated cat populations enrolled in their study have anti-AAV9 circulating antibodies that are not neutralizing and that may enhance transduction.
2. Investigators found AAV9 variants that de-target the liver and other peripheral organs down the transcription level.
3. Investigators found that screening of AAV9 variants in mice does not always predict outcomes in cats.

1. Defined the micro-RNA (miRNA) to be delivered to Sertoli cells that would effectively suppress androgen receptor (AR) production and defined and created a construct with a cell specific promoter so that the miRNA would only be produced in the targeted cell. Tested this and demonstrated effectiveness in cultured cells.
2. Engineered lentiviral particles to carry miRNAs into the cells. Further, the viral envelope of the lentivirus was modified to express a small peptide that bound to FSH receptors, which ensures that the lentivirus will “home” to FSHR-expressing Sertoli cells and, in addition, engineered the modified virus to make it non-infective to humans.
3. Tested their construct in male mice, via injection into the efferent ducts of the testes, and showed suppression of fertility for 5 months (length of the study). Investigators also showed that they did not get as much specific targeting when the construct was given IV, indicating they need more refinement of the peptide targeting mechanism.
4. Demonstrated that targeting the androgen-AR pathway with miRNA and cell-specific targeting viruses is a viable therapeutic option for development of a single-dose nonsurgical sterilant.

1. SPRASA was localized in ovaries and testes of prepuberal dogs and cats.
2. Cat and dog SPRASA reactive antisera were generated. Mice were immunized with an anti-SPRASA vaccine or control and allowed to mate.
3. Thirty-six of 42 SPRASA-immunized and all 7 control mice became pregnant with normal litter & fetus size.
4. Six SPRASA immunized mice never became pregnant. Not all sterile mice were obese, which is a side effect in other species.
5. Number of sperm and number of progressively motile sperm was lower in immunized (n=7) males, but were not significantly different than in control males, and fertility was the same.

1. Safety results show that the polyanhydride rods were well-tolerated, and implants were tolerated moderately well, in male and female mice.
2. Histology results show that the implant created a tissue/PVDF membrane interface with neovascularization and formation of lymphocytic aggregates.
3. Immune response results showed that the vaccine platform induced long-term anti-GnRH antibody titers and cell-mediated immunity for up to 7 months in male mice and up to 10 months in female mice.
4. Efficacy results showed no evidence of azoospermia in male mice or prevention of estrus and ovulation in female mice.
5. The vaccine platform can be easily “tuned” by adjusting adjuvants and vaccine antigens to optimize the immune response.

1. A recombinant mouse cytomegalovirus was generated to express rat zona pellucida 3 glycoprotein and grown to sufficient titers to inoculate rats.
2. Rats were inoculated with this virus construct or with controls (PBS, parental non-recombinant virus); ZP3 specific antibody was demonstrated (by ELISA) at 25 day intervals in rats immunized with the treatment constructs.
3. Inoculated rats delivered 2.1 to 2.8 fewer live births than control rats, but vaccination did not induce sterility.
4. There was a slight difference in litter size between two virus dose (9.3 and 9.2 pups) compared to controls (11.9 pups).
5. Ovarian morphology revealed some “disruption to granulosa cells” but no decrease in ZP.

1. The goal of this project was to develop and test antigen presenting scaffolds in order to initiate a strong and long-lasting humoral immune response against the GnRH signal peptide.
2. A variety of scaffold compositions, adjuvants conjugated to the antigen, antigen types, and techniques for incorporating the antigen into the scaffold were developed and tested.
3. In culture, a CpG adjuvant conjugated to peptide produced similar levels of IL-12 responses compared to unconjugated CpG and GnRH and, in vivo, the highest IgG1 and IgG2 responses compared to additional TLR adjuvants (poly(I:C) (TLR-3 agonist) and MPLA Toll-Like Receptors (TLR-4 agonist).
4. In vivo, the mesoporous silica rods (MSR) scaffold system for slow release of the conjugated GnRH antigen produced the highest and longest lasting GnRH antibody levels compared to other scaffolds: 1) mesoporous silica micro-rods (MSR); 2) macroporous scaffolds fabricated from poly(lactide-co-glycolide) (PLG); and 3) cryogels fabricated from alginate and vaccine systems (bolus or alum) vaccines.
5. In vivo, although high titers to GnRH were observed with a single shot and prime/boost strategy using the MVPGnRH MSR, only the prime/boost application resulted in estrus cycle alterations as monitored by vaginal lavage which resulted in the observation of varied and inconsistent estrus cycles and as well as reproductive organ size. GnRH adjuvant alone did not produce anti-GnRH titers; only GnRH antigen containing Th epitope peptides were found to elicit the desired response.

1. Made 7 constructs that elicit GnRH antibodies in mice & could (alone or in combination) be used in dogs/cats.
2. Tested constructs in mice in single injections and produced anti-GnRH antibodies (measured in serum) but no change in T levels or testes size.
3. Conclusion was that they needed higher doses or combinations, or adjuvants or vaccine in nanoparticle format.

1. Nine different GnRHR-based DNA vaccine constructs were produced and shown to express the desired gene product in vitro.
2. DNA vaccine constructs were evaluated for biological activity following single-dose administration in 3 independent mouse studies. Delivery parameters included IM or ID administration and with or without adsorption onto PLGA or magnetic nanoparticles, or co-injection with IL-15 plasmid.
3. Two DNA constructs encoding Ub-FeGnRHR induced a significant suppression of serum testosterone levels beginning in some cases at 12 weeks and lasting up to 25 weeks. A decrease in GnRHR mRNA expression in pituitaries was found in these treatment groups.
4. Mice immunized with Ub-FeGnRHR construct driven by EF1α promoter had moderate, but statistically significant, decrease in the number of fetuses in fertility trials.

1. Exosomes were obtained from the supernatant of an immortalized mouse mesenchymal stem cell line and transduced with a lentivirus carrying a kisspeptin10 ligand.
2. Minicircle expression vectors were constructed that drive the expression of a toxic ion channel gene under a strong promoter to kill transfected cells; these vectors were loaded onto the recombinant exosomes.
3. Specificity of recombinant vs. nonrecombinant loaded exosomes could not be demonstrated in cells expressing the kisspeptin receptor.

1. The genes encoding Kisspeptin (kiss1) and its receptor (kiss1r) have been identified and sequenced in the dog and compared to relevant human cDNA sequences.
2. The amino acid sequence of canine KP-10 differs at two positions from human KP-10.
3. Canine KP-10 induces an LH response which appears to be affected by stage of the reproductive cycle.
4. The kisspeptin antagonist p271 which is antagonistic in other species is not an antagonist in dogs.
5. ln vitro work with a Ca-flux technique studying single cells with the human kisspeptin receptor revealed that both hKP10 and cKP10 resulted in a Ca2+ increase. The p271 antagonist did not prevent a Ca2+ increase.

1. The publicly available putative sequence for the rat KISS1 receptor was corrected and redeposited.
2. A protein encoded by a putative dog KISS1 receptor sequence was shown to respond to kisspeptin, while proteins from two other such sequences do not.
3. A variety of technical hurdles were overcome to allow the production from bacterial cultures of a saporin-kisspeptin and a Botulinum toxin domain-kisspeptin conjugate that bind and activate KISS1 receptors at concentrations near that of the native kisspeptin peptide. Amounts sufficient for in vivo experiments in rodents were purified.
4. In cultured cells, both the saporin-kisspeptin conjugate and the Botulinum toxin domain-kisspeptin conjugate potently and selectively kill cells expressing rat KISS1 receptor over cells that do not express the receptor.
5. Injection of the saporin-kisspeptin conjugate and the Botulinum toxin domain-kisspeptin conjugate, either IV (rats) or ICV (rats and mice), causes an LH surge, showing that the proteins reach and activate the gonadal axis at the hypothalamus. The Botulinum toxin domain-kisspeptin conjugate does not appear to kill hypothalamic GnRH neurons. ICV injection of the saporin-kisspeptin conjugate is acutely toxic to rats at the dose tested. More rat studies would be necessary to determine if and how this toxicity is specifically due to ablating GnRH neurons.

1. GnRH-toxin conjugates are internalized and effectively induce apoptosis in immortalized gonadotrope cell lines with abundant GnRH receptors. This efficacy in immortalized cells does not translate to efficacy in primary pituitary cells, or in in mice.
2. Based on results from this research, efficacy of GnRH-ribosome inactivating protein conjugates is limited by endosome sequestration and degradation of the internalized toxin.
3. The efficacy of GnRH-ribosome inactivating protein conjugates (measured in vitro in immortalized gonadotropes) can be increased by encouraging endosome disruption.
4. Further work is needed to test if these toxin-conjugates, with the addition of endosome disruptors, can be effective in mice, and eventually in dogs and cats, as a single-shot sterilant.

1. The goal of the project was to test the simultaneous delivery of a ligand-targeted toxin and ligand-targeted endosome disruptor to gonadotropes. The grantee hypothesized that this would enhance the efficacy of targeted toxins and better ablate gonadotropes. The grantee used two targeting ligands (GnRH and Metastin), one endosome disruptor (Listeriolysin O), and two toxins, both of which were ribosome inactivating proteins (Gelonin and Saporin). 
2. In a rabbit reticulocyte lysate assay of in vitro protein synthesis, GnRH conjugates of Gelonin or Saporin were equally effective in inhibiting ribosomal function and protein synthesis. 
3. In addition, both a GnRH-LLO conjugate and Metastin-LLO protein effectively lysed red blood cells at nM concentrations.
4. In immortalized gonadotropes (alphaT3-1 cells), efficacy of GnRH-Gelonin and GnRH-Saporin conjugates was enhanced by the simultaneous delivery of Metastin-LLO. This effect of Metastin-LLO was dose dependent.
5. There was some efficacy in primary pituitary cells from sheep. The optimal concentrations of GnRH-Gelonin and Metastin-LLO decreased GnRH receptor messenger RNA, but failed to decrease LH-Beta mRNA. The research team suspects that they decreased the number of gonadotropes, but that those remaining gonadotropes increased their production of LH. Future studies using the sheep primary pituitary cells will aim to better address this question.

1. Designed novel bifunctional, nonpeptide molecules capable of simultaneously exhibiting potent GnRH antagonist activity and in vitro cytotoxicity exemplified by CRN-0108.
   1. CRN-0108 is a significantly more stable and potent GnRH receptor targeted cytotoxin (in vitro) than has been previously reported.
   2. Continuous two-week infusion of CRN-0108 to castrate male rats showed partial suppression of plasma LH consistent with continuous occupancy of pituitary GnRH receptors, but not consistent with gonadotroph ablation.
   3. Results suggest that gonadotrophs in vivo were resistant to the cytotoxic activity of CRN-0108, indicating that gonadotroph cell lines may not be an appropriate in vitro screening model to predict in vivo activity.
2. Developed pharmacologic criteria for demonstrating receptor mediated effects of GnRH peptide targeted cytotoxins.
   1. Demonstrated that cell lines stably transfected with GnRH receptors are more sensitive to some chemotherapeutic agents (whether receptor-targeted or not) than parental untransfected cell lines, indicating that comparison of cytotoxicity to these cell lines cannot be used to prove GnRH receptor mediated effects.
   2. Demonstrated that in vitro cytotoxicity of several GnRH peptide targeted chemotherapeutic agents and ribosomal inactivating enzymes could not be blocked by addition of excess GnRH antagonist, indicating that the effect of these agents was not receptor mediated. The absence of this control in previously published peptide GnRH targeting approaches casts doubts as to their veracity.
3. Conclusions:
   1. Targeting of gonadotroph ablation remains a potentially promising approach to non-surgical sterilization and has not been disproven by these results. A potent agent for in vivo gonadotroph ablation remains to be discovered.
   2. These results establish important pharmacological criteria for characterizing putative GnRH receptor targeted agents.
   3. These results demonstrate an improved GnRH receptor targeting approach, although the payload to be delivered still requires further optimization.

1. Authors have targeted three cells for selective ablation that are essential for reproduction and that are not renewable after ablation: primordial oocytes, spermatogonia & Sertoli cells.
2. Oocyte homing peptide linked to the toxin MT098 caused cell death in vitro but not in vivo; at 10X increased dose in vivo there was primordial oocyte cell death; authors plan to follow up with use of a more potent cytotoxin.
3. Spermatogonia targeting with the toxin MT069 resulted in extensive spermatogonial damage and significant decrease in litter size sired by treated males. Authors plan to follow up with commercial optimization of conjugating the toxin to the targeting peptide.
4. Sertoli cell targeting was changed from that used for primordial oocytes and spermatogonia, because this targeting was not effective in SCs. Authors hypothesize that this may be due to the phagocytic & endocytic functions of SCs causing the cells to rapidly engulf and internalize homing peptides. Therefore they designed peptides that bind to the FSH receptor. Peptides were tested for binding to the FSHR and in vivo efficacy of these peptides to destroy cells when conjugated to toxins. These peptides have been shown to target SCs and granulosa cells in vivo.

1. Peptides with specificity for male and female gonads conjugated to redox cycling quinones are cytotoxic for gonadal cells in vitro.
2. Multiple doses of some targeting peptide-quinone conjugates or combinations of conjugates can cause contraception in both male and female mice.
3. While showing some promise, the requirement for multiple doses administered intraperitoneally (IP) indicates low efficacy of these initial targeting peptide-quinone conjugates and the need for further refinement of the approach.

1. PI was unable to demonstrate a correlation between ApoER2 expression and selenoprotein P (SelP) dependent selenium uptake.
2. SelP may be able to non-specifically deliver selenium to cells.
3. SelP is still a viable contraceptive target, but significant mechanistic studies will be required in order to determine the uptake pathway.

1. Authors attempted to use superparamagnetic-iron oxide nanoparticles (SPIONS) encapsulated by PEGylated phospholipids bearing toxic cargo to bind to FSH receptors (FSHR) and destroy testicular Sertoli and ovarian granulosa cells in mice.
2. Cell lines reportedly expressing FSHR and CHO cells transfected with plasmids encoding mouse FSHR did not preferentially take up the SPIONS compared to nonspecific uptake into CHO cells.
3. Authors concluded that their approach of using FSH-Pep derivatized SPIONS for targeting reproductive cells in dogs and cats probably will not work since particles also enter off-target cells not expressing FSHR.

1. Four families of peptides showing specific binding were identified.
2. A number of strong binding oocyte-specific phage clones were identified, but they could not be validated as oocyte-selective by other means.
3. Immunocytochemistry for localization of phage on ovary sections did not demonstrate cell selectivity.

1. Stable cell lines expressing the alpha and beta subunits of follicle stimulating hormone (FSH) were established.
2. A modularly assembled ligand toxin conjugation system was produced and validated using FSH fragments and a Pseudomonas toxin; the toxin is expressed as a fusion protein to an “acidic velcro” peptide that facilitates conjugation using leucine zippers as heterodimerization domains.
3. Reagents for producing the ligand-toxin conjugates are available for use by other members of the scientific community.

1. Identified the best PAR inhibitor enzyme (ABT-888) in first years of work. 
2. Identified Temozolomide and Busulfan (BU) as the best (anticancer) cytotoxic agents to use with PARP sensitized cells (to induce cell death); established best dose of PAR inhibitor and BU and administered to mice. 
3. Detected bone marrow suppression in treated mice, which subsided after 6 weeks. 
4. Spermatogenesis was ablated in treated males 6 weeks after a single dose, but treated males regained fertility by 3 months. One male mouse stayed sterile for 7 months; one sired only one litter when housed with females.

1. 32 kittens (16M & 16F) were treated with 1.6 mg deslorelin acetate or a control implant within 24 hrs of birth.
2. Puberty onset was significantly delayed in treated (mean 63 wks, range 42-91 wks) versus control (mean 15.5 +/- 1.7 wks) kittens.
3. Five (2M, 3F) deslorelin treated kittens were infertile at 3 consecutive mating trials after puberty.
4. Partial (not baseline) reduction of fecal sexual hormones in first 5 weeks of life may indicate that deslorelin dose was insufficient for an irreversible effect.
5. Gonadal weight and volume were lower in treated than control males immediately after puberty and germinal epithelium and Sertoli cell numbers were diminished.
6. Gonadal weight did not differ between treated & control females, but primordial, primary & secondary follicles per mm² were significantly decreased in treated females.

1. Investigators developed a variety of capsules to implant subcutaneously with various compositions that were designed to slow release deslorelin over time and protect it from water to retard its degradation, with the goal of having an implant that would be effective for 10+ years.
2. Implants were tested in vitro and release of deslorelin was seen with addition of a DMSO carrier.
3. Implants considered the most promising based on in vitro deslorelin release were implanted in male mice, and mice were evaluated for serum testosterone and testicular shrinkage. The implants had no effect on these parameters over 12 weeks.
4. These results were interpreted as indicating that the in vitro system did not predict in vivo deslorelin release, and if there was deslorelin release in vivo it was insufficient to cause suppression of fertility.