Michelson Grants Research Findings
The Michelson Prize & Grants provides information on general positive and negative findings from research it funds. Research findings will be added to this page as data are generated at the completion of funded projects. Click on each title below to reveal more information about each project. To learn more about the principal investigators, visit Current Grantee Profiles.
Disclaimer: Projects previously funded may or may not represent current funding priorities of the Foundation.
SILENCING OF GENES ESSENTIAL FOR REPRODUCTION
Computationally designed hyperstable anti-GnRH mini-proteins , David Baker, PhD - Project in progress
The goal of this project is to computationally design mini-proteins that will bind to gonadotropin-releasing hormone (GnRH) and thereby prevent GnRH from binding to its receptor.
David Baker, PhD, Department of Biochemistry, University of Washington, Seattle, WA USA, firstname.lastname@example.org
Project in progress
Silencing of genes coding for kisspeptin and neurokinin B in hypothalamic neurons of the rat, via systemic administration of an adeno-associated viral vector linked to a homing peptide.
- Generated siRNA against rat Kiss1 and Tac2 genes that encode for kisspeptin and neurokinin B, respectively.
- Identified homing peptides to the rat hypothalamus using phage panning.
- 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.
- 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.
- Generated siRNAs that target the common coding regions of the cat and dog Kiss1 and Tac2 genes.
- Demonstrated 2 large deletions in dog Kiss1 that are present in other canids but not in raccoons, mink, skunks or felids.
- Dissen GA, Lomniczi A, Boudreau RL, Chen YH, Davidson BL, Ojeda SR. Applying gene silencing technology to contraception. Reprod Domest Anim. 2012 Dec; 47 Suppl 6: 381-386.
- Dissen GA, Lomniczi A, Boudreau RL, Chen YH, Davidson BL, Ojeda SR. Targeted gene silencing to induce permanent sterility. Reprod Domest Anim. 2012 Aug; 47 Suppl 4: 228-232.
- Dissen GA, Lomniczi A, Boudreau RL, Chen YH, Davidson BL, Ojeda SR. Applying gene silencing technology to contraception. Oral presentation. 7th International Symposium on Canine and Feline Reproduction (ISCFR), Whistler, BC, Canada. July 26, 2012. [See corresponding publication above.]
- Dissen GA, Lomniczi A, Boudreau RL, Chen YH, Davidson BL, Ojeda SR. Targeted gene silencing to induce permanent sterility. Oral presentation. 17th International Congress on Animal Reproduction (ICAR), Vancouver, BC, Canada. July 31, 2012. [See corresponding publication above.]
- Dissen GA, Lomniczi A, Chen YH, Spengler RM, Boudreau RL, Davidson BL, Ojeda SR. Suppressing GnRH activators using gene silencing. Oral presentation. 11th International Symposium on GnRH, Salzburg, Austria. February 10, 2014.
- Ojeda SR, Lomniczi A, Boudreau RL, Chen YH, Davidson BL, Dissen GA. Gene silencing technology as a tool to induce permanent sterility. Oral presentation. Alliance for Contraception in Cats & Dogs (ACC&D) 5th International Symposium on Non-Surgical Methods of Pet Population Control, Portland, OR USA. June 23, 2013.
Targeting piRNA/endo-siRNA pathways for the control of companion animal fertility , Jonathan LaMarre, DVM, PhD - Project completed
Inhibit small RNAs that are critical for meiosis in dog oocytes and spermatogonia in tissues obtained from animals at routine neutering
Jonathan LaMarre, DVM, PhD, Department of Biomedical Sciences, University of Guelph, Guelph, Canada, email@example.com
- Identified and cloned canine and feline PIWIL1 and PIWIL2 and expressed as recombinant proteins in mammalian cells.
- Identified differential patterns of PIWIL1 and PIWIL2 expression in immature (lower) vs. mature (higher) male dogs and cats.
- Confirmed and expanded basic knowledge of piRNA/PIWI pathway involved in male gametogenesis in dogs and cats.
- Developed new, improved methods and protocols to allow for longer in vitro cultivation of dog and cat male gonadal explants.
- Stalker L, Russell SJ, Co C, Foster RA, LaMarre J. PIWIL1 is expressed in the canine testis, increases with sexual maturity, and binds small RNAs. Biol Reprod. 2016 Jan 1; 94(1): 17.
- LaMarre J. Emerging Strategies for Non-Surgical Control of Fertility in Pet Species. Oral presentation. Association of Animal Shelter Administrators of Ontario Conference, Cambridge, Ontario, Canada. September 26, 2014.
- Stalker L, Backx A, White K, Molledo G, LaMarre J. Investigation of the PIWI pathway in non-model organisms: challenges and complexities. Poster presentation. 44th Southern Ontario Reproductive Biology Meeting, Kingston Ontario, Canada. May 13, 2016.
Engineering viral vectors to target the cat hypothalamus with sterilizing molecules , Sergio Ojeda, DVM - Project completed
Using AAV Barcode-Seq to engineer an AAV that specifically homes to cat hypothalamic neurons
Sergio Ojeda, DVM, Oregon Health & Science University School of Medicine, Beaverton, OR USA, firstname.lastname@example.org
- 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.
- Investigators found AAV9 variants that de-target the liver and other peripheral organs down the transcription level.
- Investigators found that screening of AAV9 variants in mice does not always predict outcomes in cats.
- Dissen G, Adachi K, Lomniczi A, Chatkupt T, Davidson B, Nakai H, Ojeda S. Engineering a gene silencing viral construct that targets the cat hypothalamus to induce permanent sterility: An update. Reprod Dom Anim. 2016; 51(Suppl. 3): 1-5.
- Dissen GA. Inducing sterility using gene therapy in cats and dogs. Seminar. University of Saskatchewan, Saskatoon, SK, Canada. December 15, 2015.
- Dissen GA, Lomniczi A, Adachi K, Chatkupt T, Davidson BL, Nakai H, Ojeda SR. Engineering a gene silencing viral construct that targets the cat hypothalamus to induce permanent sterility. Oral presentation. 8th International Symposium on Canine and Feline Reproduction (ISCFR), Paris, France. June 24, 2016.
Using microRNA technology to inhibit expression of androgen receptor protein in the testes of male mice, interfering with testosterone’s ability to support sperm production.
- 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.
- 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.
- 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.
- 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.
- Darbey A, Smith LB. Deliverable transgenics & gene therapy possibilities for the testes. Mol Cell Endocrinol. 2017 Dec 1. pii: S0303-7207(17)30609-3. doi: 10.1016/j.mce.2017.11.023. [Epub ahead of print]
- Smith LB, O’Shaughnessy PJ, Rebourcet D. Cell-specific ablation in the testis: What have we learned? Andrology. 2015; 3(6): 1035-1049.
- Willems A, Roesl C, Mitchell RT, Milne L, Jeffery N, Smith S, Verhoeven G, Brown P, Smith LB. Sertoli cell androgen receptor signalling in adulthood is essential for post-meiotic germ cell development. Mol Reprod Dev. 2015 Sept; 82(9): 626-627.
- Darbey A, Roesl C, Jeffery N, Smith SE, O’Hara L, Cruickshanks L, Mitchell RT, Brown P, Smith LB. Tissue repair in the testis through viral gene therapy. Poster & Oral presentation. 19th European Testis Workshop, Saint-Malo, France. June 11-15, 2016.
- Roesl C. Sterilisation via disruption of androgen signalling – a novel gene therapy approach. Oral presentation. Institute of Fundamental Sciences at Massey University, Palmerston North, New Zealand. May 7, 2015.
- Roesl C, Jefferey N, Smith SE, Cruickshanks L, Mitchell RT, Brown P, Smith LB. Single-injection sterility via lentiviral-mediated suppression of Androgen Receptor in Sertoli cells. Oral presentation. 8th International Symposium on Canine and Feline Reproduction (ISCFR), Paris, France. June 24, 2016.
- Roesl C, Jefferey N, Smith SE, Milne L, Brown P, Smith LB. Development of a single-injection non-surgical sterilant via modification of Measles Virus pseudotyped particles. Poster presentation. Gordon Research Conference: Cells and Viruses, Girona, Spain. June 21-26, 2015.
- Roesl C, Jefferey N, Smith SE, Milne L, Brown P, Smith LB. Development of a single-injection non-surgical sterilant via modification of Measles Virus pseudotyped particles. Poster presentation. British Society for Andrology BES Conference, Edinburgh, UK. November 2-4, 2015.
- Smith LB. Sterilisation via disruption of androgen signalling – a novel gene therapy approach. Oral presentation. Laval University, Quebec City, QC, Canada. September 11, 2015.
Translation of an androgen-miRNA sterilant: pre-clinical validation & a clinical trial in cats and dogs , Lee Smith, PhD - Project in progress
In their first, completed project, investigators used microRNA technology to inhibit expression of androgen receptor protein in the testes of male mice, thereby interfering with testosterone’s ability to support sperm production. Investigators showed efficacy in targeting the androgen-AR pathway and demonstrated induction of sterility in male mice for 5 months. In this new project, investigators will further refine their single-dose sterilant construct before initiating a clinical trial in cats and dogs.
Project in progress
Induction of infertility/sterility in male and female mice immunized against Sperm Reactive Protein with Antisperm Antibodies (SPRASA).
Larry Chamley, PhD, University of Auckland, Auckland, New Zealand, email@example.com
- SPRASA was localized in ovaries and testes of prepuberal dogs and cats.
- Cat and dog SPRASA reactive antisera were generated. Mice were immunized with an anti-SPRASA vaccine or control and allowed to mate.
- Thirty-six of 42 SPRASA-immunized and all 7 control mice became pregnant with normal litter & fetus size.
- Six SPRASA immunized mice never became pregnant. Not all sterile mice were obese, which is a side effect in other species.
- 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.
- Wagner A, Holland OJ, Tong M, Shelling AN, Chamley LW. The role of SPRASA in female fertility. Reprod Sci. 2015 Apr; 22(4): 452-461.
- Holland O, Tong M, Chamley L. Expression of a novel immunocontraceptive target in the gonads of cats and dogs. Poster presentation. 12th Congress of the International Society for Immunology of Reproduction, Boston, MA USA. May 28, 2013.
Development of a vaccine delivery device that will maintain life-long high titers of anti-GnRH antibodies , Douglas E. Jones, MS, VMD, PhD - Project completed
Immunization of mice against GnRH using extended release of antigen from bioerodable beads and maintaining high titers using immune-regulated release of antigen from a cyto-exclusive implant.
Douglas E. Jones, VMD, PhD, Iowa State University, Ames, IA USA, firstname.lastname@example.org
- Safety results show that the polyanhydride rods were well-tolerated, and implants were tolerated moderately well, in male and female mice.
- Histology results show that the implant created a tissue/PVDF membrane interface with neovascularization and formation of lymphocytic aggregates.
- 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.
- Efficacy results showed no evidence of azoospermia in male mice or prevention of estrus and ovulation in female mice.
- The vaccine platform can be easily “tuned” by adjusting adjuvants and vaccine antigens to optimize the immune response.
- Schaut RG, Brewer MT, Hostetter JM, Mendoza K, Vela-Ramirez JE, Kelly SM, Jackman JK, Dell’Anna G, Howard JM, Narasimhan B, Zhou W, Jones DE. A single dose polyanhydride-based vaccine platform promotes and maintains anti-GnRH antibody titers. Vaccine. 2018 Feb 8;36(7):1016-1023.
- Schaut RG, Brewer MT, Mendoza K, Jackman J, Narasimhan B, Jones DE. A polyanhydride-based implantable single dose vaccine platform for long-term immunity. Vaccine. 2018 Feb 8;36(7):1024-1025.
- Jones DE, Brewer M, Li J, Bockenstedt M, Ramirez J, Narasimhan B, Mallapragada S. Immunization against GnRH for non-surgical sterilization. Oral presentation. 11th International Symposium on GnRH, Salzburg, Austria. February 10, 2014.
- Mendoza KA, Brewer M, Narasimhan B, Ramirez J, Jones DE. An in vitro study developing a vaccine delivery device that will maintain life-long titers of anti-GnRH antibodies. Poster presentation. Merial-NIH National Veterinary Scholars Symposium, Cornell University CVM, Ithaca, NY USA. July 31-August 3, 2014.
Contraception in companion animals using a recombinant viral vector , Megan Lloyd, PhD - Project - Project completed
Immunization of rats against three zona pellucida antigens in a mouse cytomegalovirus vector.
Megan Lloyd, PhD, University of Western Australia, Crawley WA, Australia, email@example.com
- A recombinant mouse cytomegalovirus was generated to express rat zona pellucida 3 glycoprotein and grown to sufficient titers to inoculate rats.
- 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.
- Inoculated rats delivered 2.1 to 2.8 fewer live births than control rats, but vaccination did not induce sterility.
- There was a slight difference in litter size between two virus dose (9.3 and 9.2 pups) compared to controls (11.9 pups).
- Ovarian morphology revealed some “disruption to granulosa cells” but no decrease in ZP.
Infection-mimicking biomaterials for vaccination against gonadotropin releasing hormone (GnRH) , David Mooney, PhD - Project in progress
Vaccination against GnRH using infection-mimicking biomaterial scaffolds
David Mooney, PhD, Harvard University, Cambridge, MA USA, firstname.lastname@example.org
Project in progress
- Li WA, Lu BY, Gu L, Choi Y, Kim J, Mooney DJ. The effect of surface modification of mesoporous silica micro-rod scaffold on immune cell activation and infiltration. Biomaterials. 2016 Mar; 83: 249-56.
Developing herpesvirus vectors to induce long-term immunity to reproductive hormones and hormone receptors , Michael Munks, PhD - Project completed
Immunization of cats against GnRH, ZP antigens and gonadotropin receptors using a feline herpesvirus vector.
Michael Munks, PhD, Oregon Health & Science University, Portland, OR USA, email@example.com
- Munks MW. Progress in development of immunocontraceptive vaccines for permanent non-surgical sterilization of cats and dogs. Reprod Domest Anim. 2012 Aug; 47 Suppl 4: 223-227.
- Munks MW, Montoya AM, Pywell CM, Talmage G, Forssen A, Campbell TL, Dodge DD, Kappler JW, Marrack P. The domestic cat antibody response to feline herpesvirus-1 increases with age [published online May 4 2017]. Vet Immunol Immunopathol. 2017.
- Munks MW. Use of recombinant feline herpesvirus-1 (FHV-1) as a vaccine vector for cat immunocontraception. Oral presentation. 8th International Symposium on Canine and Feline Reproduction (ISCFR), Paris, France. June 24, 2016.
- Munks MW. Use of feline herpesvirus-1 (FHV-1) as a cat contraceptive vaccine vector. Oral presentation. Alliance for Contraception in Cats & Dogs (ACC&D) 5th International Symposium on Non-Surgical Methods of Pet Population Control, Portland, OR USA. June 21, 2013.
- Munks MW. Progress in development of immunocontraceptive vaccines for permanent non-surgical sterilization of cats and dogs. Oral presentation. 17th International Congress on Animal Reproduction (ICAR), Vancouver, BC, Canada. July 31, 2012. [See corresponding publication above.]
Self-boosting pathogen-like particle multi-antigen vaccine for female immunosterilization , David Putnam, PhD - Project completed
Self boosting outer membrane vesicle (OMV) with polymeric microparticles (PLGA) multi-antigen vaccine for female immunosterilization in rats
David Putnam, PhD, Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA, firstname.lastname@example.org
Phage-GnRH constructs and their mimics for immunocontraception of cats and dogs , Tatiana I. Samoylova, PhD - Project completed
Use of phage particles and their mimics bearing ~4000 copies of GnRH in a single dose anti-GnRH vaccine to induce lifetime immunocontraception in mice.
- Made 7 constructs that elicit GnRH antibodies in mice & could (alone or in combination) be used in dogs/cats.
- Tested constructs in mice in single injections and produced anti-GnRH antibodies (measured in serum) but no change in T levels or testes size.
- Conclusion was that they needed higher doses or combinations, or adjuvants or vaccine in nanoparticle format.
- Napier ID, Samoylov AM, Morrison N, Samoylova T. Molecular cloning of CHO-K1 cells expressing canine gonadotropin-releasing hormone receptor. Abstract. Society for the Study of Reproduction 45th Annual Meeting and Ovarian Workshop, State College PA USA. August 12-15, 2012.
- Samoylov A, Cochran A, Schemera B, Kutzler M, Donovan C, Petrenko V, Bartol F, Samoylova T. Humoral immune responses against gonadotropin releasing hormone elicited by immunization with phage-peptide constructs obtained via phage display. Journal of Biotechnology. 2015 Dec 20; 216: 20-28.
- Samoylov A, Napier I, Morrison N, Cox N, Samoylova T. Molecular cloning, sequencing and characterization of feline gonadotropin releasing hormone receptor. Abstract. Society for the Study of Reproduction 46th Annual Meeting, Montreal, QC, Canada. July 22-25, 2013.
- Samoylov AM, Napier ID, Morrison NE, Cox NN, Samoylova TI. Felis catus gonadotropin-releasing hormone receptor (GnRHR) mRNA, complete cds. GenBank Accession Number JX157624.
- Samoylov AM, Napier ID, Morrison NE, Martin DR, Cox NR, Samoylova TI. Molecular cloning, sequencing, and distribution of feline GnRH receptor (GnRHR) and resequencing of canine GnRHR. Theriogenology. 2015 Jan 15; 83(2): 266-275.
- Samoylova T, Samoylov AM, Cochran AM, Schemera B, Kutzler M, Donovan C, Petrenko VA, Cox NR, Bartol FF. Phage-peptide constructs for elucidation of humoral immune responses against gonadotropin releasing hormone. Abstract. Society for the Study of Reproduction Annual Meeting, San Juan, Puerto Rico. June 18-22, 2015.
- Samoylova TI, Braden TD, Spencer JA, Bartol FF. Immunocontraception: Filamentous bacteriophage as a platform for vaccine development. Curr Med Chem. 2017 Oct; 24(35): 3907–3920.
- Samoylova T. Filamentous phage as a platform for development of contraceptive vaccines for animals. Oral presentation. Alliance for Contraception in Cats & Dogs (ACC&D) 5th International Symposium on Non-Surgical Methods of Pet Population Control, Portland, OR USA. June 21, 2013.
- Samoylova T. Filamentous phage as platform for vaccine design and delivery. Oral presentation. 9th World Drug Delivery Summit, New Orleans, LA USA. June 30‐July 2, 2016.
Ablation of pituitary gonadotropes by DNA vaccine targeting GnRH receptor: Proof of principle study in mice , Tatiana I. Samoylova, PhD - Project completed
Create a slow release DNA vaccine against the GnRH-R that will ablate pituitary and reproductive function in male mice
- Nine different GnRHR-based DNA vaccine constructs were produced and shown to express the desired gene product in vitro.
- 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.
- 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.
- Mice immunized with Ub-FeGnRHR construct driven by EF1α promoter had moderate, but statistically significant, decrease in the number of fetuses in fertility trials.
- Napier ID, Anani TB, Yeh BJ, Samoylov AM, David AE, Samoylova TI. Superparamagnetic iron oxide nanoparticles for delivery of DNA-based contraceptive vaccines for feral cats. Abstract. Society for the Study of Reproduction Annual Meeting, San Juan, Puerto Rico. June 18-22, 2015.
- Samoylov A, Napier I, Cochran A, Schemera B, Morrison N, Wright J, Cattley R, Samoylova T. Intradermal delivery of DNA vaccines targeting GnRHR. Poster. 9th World Drug Delivery Summit, New Orleans, LA. June 30‐July 2, 2016.
A vectored GnRH contraceptive vaccine to control dog and cat overpopulation , Kent R. Van Kampen, DVM, PhD - Project completed
Immunization of mice, cats and dogs against GnRH using an adenovirus vector expressing multiple GnRH repeats.
Kent Van Kampen, DVM, PhD, Vaxin Inc., Gaithersburg, MD USA, email@example.com
TARGETED DELIVERY OF CYTOTOXINS
To Hypothalamic Neurons
Targeted ablation of GnRH neurons: proof of concept study in mice , Meenakshi Alreja, PhD - Project completed
Targeted ablation of GnRH neurons with a kisspeptin-toxin conjugate: proof of concept in mice.
Meenakshi Alreja, PhD, Yale University School of Medicine, New Haven, CT USA, firstname.lastname@example.org
Over-expression of the novel Rfamide gonadotropin-inhibitory hormone: Proof of concept study in rats , George Bentley, PhD - Project completed
Over-expression of the novel Rfamide gonadotropin-inhibitory hormone: Proof of concept study in rats
George Bentley, PhD, University of California, Berkeley, Berkeley CA USA, email@example.com
- First demonstration of GnIH function in male and female feline gonads – inhibition of steroid production.
- Successfully constructed an AAV-GnIH vector and demonstrated in vitro activity (increased RFRP expression).
- AAV-GnIH single-dose administration to male rats conferred a significant, transient increase in body weight over the first 2 months post-administration.
- 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.
Ablation of hypothalamic GnRH neurons using targeted, cytotoxic exosomes: Proof of concept study in mice , Colin E. Bishop, PhD - Project completed
Ablation of hypothalamic GnRH neurons using targeted, cytotoxic exosomes: Proof of concept study in mice.
Colin E. Bishop, PhD, Wake Forest Baptist Medical Center, Winston-Salem, NC USA, firstname.lastname@example.org
- Exosomes were obtained from the supernatant of an immortalized mouse mesenchymal stem cell line and transduced with a lentivirus carrying a kisspeptin10 ligand.
- 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.
- Specificity of recombinant vs. nonrecombinant loaded exosomes could not be demonstrated in cells expressing the kisspeptin receptor.
Kisspeptin: The endocrinological gatekeeper to reproductive function. A realistic target for non-surgical contraceptive in the dog , A.C. Schaefers-Okkens, DVM, PhD - Project completed
Determine the effect of canine kisspeptin (KP) and/or the antagonist KP 234-penetratin on the pituitary-gonadal axis in different phases of the cycle and anestrus in intact bitches and castrated dogs.
Auke Schaefers-Okkens, DVM, PhD, Faculty of Veterinary Medicine, University of Utrecht, The Netherlands, A.C.Schaefersemail@example.com
- The genes encoding Kisspeptin (kiss1) and its receptor (kiss1r) have been identified and sequenced in the dog and compared to relevant human cDNA sequences.
- The amino acid sequence of canine KP-10 differs at two positions from human KP-10.
- Canine KP-10 induces an LH response which appears to be affected by stage of the reproductive cycle.
- The kisspeptin antagonist p271 which is antagonistic in other species is not an antagonist in dogs.
- 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.
- Albers-Wolthers CHJ, de Gier J, Rutten VPMG, van Kooten PJS, Leegwater PAJ, Schaefers-Okkens AC, Kooistra HS. The effects of kisspeptin agonist canine KP-10 and kisspeptin antagonist p271 on plasma LH concentrations during different stages of the estrous cycle and anestrus in the bitch. Theriogenology. 2016 Jul 15; 86(2): 589–595.
- Schaefers-Okkens AC, Albers-Wolthers CHJ, De Gier J, Kooistra HS. Kisspeptin, a realistic target for non-surgical contraception in the dog. Oral presentation. Annual Congress of the European Veterinary Society for Small Animal Reproduction (EVSSAR), Wroclaw, Poland. September 26, 2014.
- Schaefers-Okkens AC, Albers-Wolthers CHJ, De Gier J, Kooistra HS. Kisspeptin, a realistic target for non-surgical contraception in the dog. Oral presentation. 11th International Symposium on GnRH, Salzburg, Austria. February 10, 2014.
Targeted ablation of GnRH neurons for nonsurgical sterilization , R. Scott Struthers, PhD - Project completed
Targeted ablation of GnRH neurons for nonsurgical sterilization.
- The publicly available putative sequence for the rat KISS1 receptor was corrected and redeposited.
- 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.
- 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.
- 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.
- 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.
- Struthers RS, Zhu F, Kusnetzow AK, Betz SF. HPG-targeted toxins for nonsurgical sterilization of cats and dogs. Oral presentation. 11th International Symposium on GnRH, Salzburg, Austria. February 10, 2014.
To Gonadotropes of the Anterior Pituitary
Prepubertal administration of GnRH agonists in domestic cats , Cristina Gobello, MV, DVM, DECAR - Project completed
Prevention/delay of onset of puberty in cats given a GnRH analogue in the prepubertal period.
- 32 kittens (16M & 16F) were treated with 1.6 mg deslorelin acetate or a control implant within 24 hrs of birth.
- Puberty onset was significantly delayed in treated (mean 63 wks, range 42-91 wks) versus control (mean 15.5 +/- 1.7 wks) kittens.
- Five (2M, 3F) deslorelin treated kittens were infertile at 3 consecutive mating trials after puberty.
- 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.
- Gonadal weight and volume were lower in treated than control males immediately after puberty and germinal epithelium and Sertoli cell numbers were diminished.
- Gonadal weight did not differ between treated & control females, but primordial, primary & secondary follicles per mm2 were significantly decreased in treated females.
- Carranza A, Faya M, Fernandez P, Barbeito C, Gobello C. Histologic effect of a postnatal slow-release GnRH agonist on feline gonads. Theriogenology. 2015 May; 83(8): 1368–1372.
- Carranza A, Faya M, Merlo ML, Batista P, Gobello C. Effect of GnRH analogs in postnatal domestic cats. Theriogenology. 2014 Jul 1; 82(1): 138-143.
- Carranza A, Merlo ML, Gobello C, Barbeito C. Histological effect of a GnRH agonist on the postnatal feline testis. Poster presentation. Congreso de la Sociedad Latinoamericana de Reproduccion (SOLARA), Buenos Aires, Argentina. March 25-28, 2015.
- Carranza A, Faya M, Batista P, Fernandez P, Gobello C, Barbeito C. Histological effect of a GnRH agonist on the postnatal feline ovary: A preliminary report. Oral presentation. Il Simposio Latinoamericano de Reproduccion Animal, Universidad de Chile, Santiago, Chile. November 13-14, 2014.
- Carranza A, de la Sota P, Diaz JD, Blanco P, Corrada Y, Gobello C. Effects of prepubertal GnRH agonist administration in domestic cats: Preliminary results. Oral presentation. 1st International Conference on Dog Population Management, York, UK. September 6, 2012.
Prepubertal administration of a long term release GnRH agonist in domestic dogs: A Pilot Study , Cristina Gobello, MV, DVM, DECAR - Project in progress
Prepubertal administration of GnRH agonists in domestic dogs.
Project in progress
- Faya M, Priotto M, Marchetti C, Dela Sota P, Gobello C. Neonatal administration of deslorelin acetate in domestic dogs: Preliminary results. Oral presentation. 8th International Symposium on Canine and Feline Reproduction (ISCFR), Paris, France. June 24, 2016.
Electrospun delivery to enhance the effectiveness of anti-fertility strategies , John Lannutti, PhD - Project completed
Long-term delivery of GnRH agonists from electrospun nanofiber delivery vehicles to suppress reproduction in mice and dogs.
John Lannutti, PhD, Ohio State University, Columbus, OH USA, firstname.lastname@example.org
- 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.
- Implants were tested in vitro and release of deslorelin was seen with addition of a DMSO carrier.
- 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.
- 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.
- Reese CM, Fuchs SE, Chaparro FJ, Carney SA, da Silva MC, Lannutti JJ. Nanofiber Capsules as Contraceptive Vehicles for Long Term Release. Poster presentation. 2015 OSU Materials Week, The Ohio State University, Columbus, OH. May 15, 2015.
Increasing the circulating half-life of GnRH-RIP conjugates to improve in vivo efficacy , Benjamin Renquist, PhD - Project completed
Increase stability and half life of GnRH-RIP conjugates to enhance cytotoxic destruction of pituitary gonadotrophs.
- 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.
- Based on results from this research, efficacy of GnRH-ribosome inactivating protein conjugates is limited by endosome sequestration and degradation of the internalized toxin.
- The efficacy of GnRH-ribosome inactivating protein conjugates (measured in vitro in immortalized gonadotropes) can be increased by encouraging endosome disruption.
- 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.
Enhancing the toxicity of GnRH- and bivalent-targeted RIP conjugates to induce sterility , Benjamin Renquist, PhD - Project in progress
Authors will continue their investigation of how to safely destroy gonadotropes, which are cells in the pituitary gland that produce hormones required for sperm production and ovulation in males and females, respectively. Complete gonadotrope destruction results in sterility. Because all gonadotropes express GnRH receptors, the Renquist lab is delivering toxins to gonadotropes by attaching toxins to GnRH. However, initial findings by this research group have shown that gonadotropes are resistant to targeted toxins. In this project, the researchers will focus on enhancing the efficacy of internalized toxins. Authors envision having an optimized cytotoxin for application in mice by the end of the study.
Project in progress
Novel toxin conjugates for nonsurgical sterilization via gonadotroph ablation , R. Scott Struthers, PhD - Project completed
Ablate pituitary gonadotrophs in rats with a small molecule GnRH antagonist conjugated to a potent small molecule toxin.
- Designed novel bifunctional, nonpeptide molecules capable of simultaneously exhibiting potent GnRH antagonist activity and in vitro cytotoxicity exemplified by CRN-0108.
- CRN-0108 is a significantly more stable and potent GnRH receptor targeted cytotoxin (in vitro) than has been previously reported.
- 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.
- 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.
- Developed pharmacologic criteria for demonstrating receptor mediated effects of GnRH peptide targeted cytotoxins.
- 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.
- 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.
- 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.
- These results establish important pharmacological criteria for characterizing putative GnRH receptor targeted agents.
- These results demonstrate an improved GnRH receptor targeting approach, although the payload to be delivered still requires further optimization.
- Struthers RS. Gonadotropin-releasing hormone targeting for gonadotroph ablation: an approach to non-surgical sterilization. Reprod Domest Anim. 2012 Aug; 47 Suppl 4: 233-238.
- Betz SF, Struthers S, Zhu Y, Kusnetzow AK. Approaches to gonadotroph ablation using GnRH receptor targeted toxins. Oral presentation. 8th International Symposium on Canine and Feline Reproduction (ISCFR), Paris, France. June 24, 2016.
- Struthers RS. Hypothalamic pituitary targeted approaches for non-surgical sterilization of cats and dogs: Using a GnRH antagonist to deliver a toxin to gonadotrophs. Oral presentation. Alliance for Contraception in Cats & Dogs (ACC&D) 5th International Symposium on Non-Surgical Methods of Pet Population Control, Portland, OR USA. June 22, 2013.
- Struthers RS. Gonadotropin-releasing hormone targeting for gonadotroph ablation: an approach to non-surgical sterilization. Oral presentation. 17th International Congress on Animal Reproduction (ICAR), Vancouver, Canada. July 31, 2012. [See corresponding publication above.]
To the Gonads
Development of a humane non-surgical sterilization method for domestic animals , R. John Aitken, ScD, FRSE - Project completed
Destroy primordial follicles and spermatogonial stem cells in mouse gonads by (i) identifying specific binding peptides using phage panning, (ii) couple peptides with cytotoxin, and (iii) subject target cells to toxin to kill them.
- 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.
- 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.
- 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.
- 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.
Alkylated FSH peptides as mediators of germ cell depletion , R. John Aitken, ScD, FRSE - Project completed
Alkylated FSH peptides as mediators of germ cell depletion.
- Peptides with specificity for male and female gonads conjugated to redox cycling quinones are cytotoxic for gonadal cells in vitro.
- Multiple doses of some targeting peptide-quinone conjugates or combinations of conjugates can cause contraception in both male and female mice.
- 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.
Targeting selenoprotein P for male contraception in mammals , Paul R. Copeland, PhD - Project completed
Characterize the testis-specific isoform of the receptor for selenoprotein P, and develop a cell culture system expressing that isoform to screen inhibitors as potential male contraceptives.
Paul R. Copeland, PhD, UMDNJ Robert Wood Johnson Medical School, Piscataway, NJ USA, Current email: email@example.com
- PI was unable to demonstrate a correlation between ApoER2 expression and selenoprotein P (SelP) dependent selenium uptake.
- SelP may be able to non-specifically deliver selenium to cells.
- SelP is still a viable contraceptive target, but significant mechanistic studies will be required in order to determine the uptake pathway.
Single treatment with AAV9 Mullerian Inhibiting Substance as an ideal permanent contraceptive , Patricia Donahoe, MD - Project in progress
Using gene therapy expressing Mullerian Inhibiting Substance to induce sterility in mice by blocking follicle recruitment and testosterone production
Patricia Donahoe, MD, Massachusetts General Hospital, Boston, MA USA, firstname.lastname@example.org
Project in progress
- Kano M, Sosulski AE, Zhang L, Saatcioglu HD, Wang D, Nagykery N, Sabatini ME, Gao G, Donahoe PK, Pépin D. AMH/MIS as a contraceptive that protects the ovarian reserve during chemotherapy. Proc Natl Acad Sci U S A. 2017 Jan 30; Epub ahead of print.
- Pepin D, Kano M, Sosulski A, Zhang L, Nicolaou F, Nagykery N, Wang D, Gao G, Donahoe PK. Gene therapy with Mullerian inhibiting substance as a female dog and cat contraceptive with lifetime suppression of fertility. Oral presentation. 8th International Symposium on Canine and Feline Reproduction (ISCFR), Paris, France. June 24, 2016.
Development of targeted nanoparticles as non-surgical sterilizing agents: proof of concept study in mice , George L. Gerton, PhD - Project completed
Design and synthesize superparamagnetic iron-oxide nanoparticles (SPIONS) that, when injected IV, IP or IT, will home to the gonads and kill cells.
George L. Gerton, PhD, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA USA, email@example.com
- 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.
- 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.
- 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.
Primary oocyte ablation by peptide-toxin conjugates and adeno-associated virus transductional and transcriptional targeting , John C. Herr, PhD - Project completed
Ablate primary oocytes in mice by peptides identified by phage planning linked to toxins
John C. Herr, PhD, School of Medicine, University of Virginia, Charlottesville, VA USA
- Four families of peptides showing specific binding were identified.
- A number of strong binding oocyte-specific phage clones were identified, but they could not be validated as oocyte-selective by other means.
- Immunocytochemistry for localization of phage on ovary sections did not demonstrate cell selectivity.
FSH receptor ligand-cytotoxin conjugates for permanent chemosterilization , William W. Ja, PhD - Project completed
Production & purification of multiple conjugates of exotoxin A and various FSH fragments tested for induction of cytotoxicity in cell cultures of mouse Sertoli and granulosa cells expressing FSH-R. Determine FSH-R expression in the mouse and the dog.
William Ja, PhD, Scripps Research Institute, Jupiter, FL USA, firstname.lastname@example.org
- Stable cell lines expressing the alpha and beta subunits of follicle stimulating hormone (FSH) were established.
- 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.
- Reagents for producing the ligand-toxin conjugates are available for use by other members of the scientific community.
Targeting poly(ADP-ribose) metabolism for development of a nonsurgical sterilant , Ralph G. Meyer, PhD - Project completed
Permanently ablate testicular germ cells in cell culture and in vivo in the mouse by inhibiting the PAR pathway (that mediates repair of DNA breaks) while administering an alkylating anti-cancer drug that is known to induce DNA strand breaks.
Ralph Meyer, PhD, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA USA, Current email: email@example.com
- Identified the best PAR inhibitor enzyme (ABT-888) in first years of work.
- 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.
- Detected bone marrow suppression in treated mice, which subsided after 6 weeks.
- 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.
Targeting the luteinizing hormone receptor to induce infertility , Prema Narayan, PhD - Project completed
Targeting the luteinizing hormone receptor to induce infertility
Prema Narayan, PhD, Southern Illinois University, Carbondale, IL USA, firstname.lastname@example.org