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
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.
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.
2. 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.
2. 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.]
3. 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.
4. 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
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.
2. 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 in progress
2. 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.
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.
2. 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.
2. 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.
3. 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.
4. 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.
5. 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
Induction of infertility/sterility in male and female mice immunized against Sperm Reactive Protein with Antisperm Antibodies (SPRASA).
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.
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.
Contraception in companion animals using a recombinant viral vector, Megan Lloyd, PhD - Project completed
Immunization of rats against three zona pellucida antigens in a mouse cytomegalovirus vector.
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.
Infection-mimicking biomaterials for vaccination against gonadotropin releasing hormone (GnRH), David Mooney, PhD - Project in progress
Vaccination against GnRH using infection-mimicking biomaterial scaffolds
Developing herpesvirus vectors to induce long-term immunity to reproductive hormones and hormone receptors, Michael Munks, PhD - Project in progress
Immunization of cats against GnRH, ZP antigens and gonadotropin receptors using a feline herpesvirus vector.
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
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.
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.
2. 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.
3. 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.
4. Samoylov AM, Napier ID, Morrison NE, Cox NN, Samoylova TI. Felis catus gonadotropin-releasing hormone receptor (GnRHR) mRNA, complete cds. GenBank Accession Number JX157624.
5. 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.
6. 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.
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
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.
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.
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.
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
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.
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.
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.
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
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.
Targeted ablation of GnRH neurons for nonsurgical sterilization, R. Scott Struthers, PhD - Project completed
Targeted ablation of GnRH neurons for nonsurgical sterilization.
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.
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.
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.
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.
Electrospun delivery to enhance the effectiveness of anti-fertility strategies, John Lannutti, PhD - Project in progress
Long-term delivery of GnRH agonists from electrospun nanofiber delivery vehicles to suppress reproduction in mice and dogs
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.
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.
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.
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.
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.
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 havbe 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 in progress
Alkylated FSH peptides as mediators of germ cell depletion.
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.
2. Sel P 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.
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
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.
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.
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
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.
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.
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.
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.
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.
Targeting the luteinizing hormone receptor to induce infertility, Prema Narayan, PhD - Project completed
Targeting the luteinizing hormone receptor to induce infertility