Gene Therapy and Immunocontraception

This lecture was pretty interesting! Probably my favourite lecture from this unit :D

Why immunocontraception?

Immunocontraception has been proposed as a potential solution to dealing with pests. For example, mouse plagues are a problem in grain-growing regions of Australia. In the past, they used to be dealt with using a poison called strychnine, but the use of strychnine has since been banned due to the risk of contamination. Currently mouse populations are monitored by using canola cards (the more chewed up the card, the bigger the problem you have), and controlled by using zinc phosphide and bromodiolone. In the USA, wild horse populations are problematic, and control measures may be needed.

Autoimmunity and infertility

Studies have shown that antibodies against the zona pellucida (ZP) are associated with infertility. The zona pellucida is a glycoprotein layer that surrounds the oocyte, providing protection from polyspermy. In mice, the zona pellucida is formed from three genes: ZP1, ZP2 and ZP3. ZP1 is associated with structural integrity, and loss of structural integrity occurs if ZP1 is knocked out. ZP2 and ZP3 alternate to form chains that make up the bulk of the zona pellucida. If ZP2 is knocked out, the early ZP is thin, and later on it is absent entirely. If ZP3 is knocked out, the ZP fails to form. In both ZP2 and ZP3 knockout, infertility results.

Studies have shown that ZP3 from a different species can be used as an "infertility vaccine" for large animals that can be vaccinated individually, such as horses. (It's a tad harder to vaccinate individual mice during a mouse plague). To address this problem, virally vectored immunocontraception was explored instead. The idea behind this process is that the DNA encoding reproductive proteins can be inserted into a virus, which can then be used to infect mice. When mice develop an immune response against the virus, they will also develop an immune response against the protein.

rMCMV-mZP3

Many immunocontraception studies examined the use of ZP3 as an antigen. This is because monoclonal antibodies to ZP3 have been shown to prevent oocyte fertilisation (both in vivo and in vitro), and immunisation with ZP3 peptide conjugated to Keyhole limpet haemocyanin also causes infertility. The vector used was MCMV (mouse cytomegalovirus), as it has a large genome (~235kb), is a natural pathogen of mice in Australia, and mice can be infected by multiple strains. It is also a persistent, latent infection, so it'll hang around for a while. MCMV containing ZP3 DNA is also known as rMCMV-mZP3.

rMCMV-mZP3 was found to induce infertility over the long term. By day 7, degradation of the zona pellucida was evident, by day 21, primary follicles had decreased, secondary follicle morphology was abnormal and tertiary follicles were absent, and by day 100, primary and secondary follicles were markedly reduced, there were no tertiary follicles, and there were evident changes to the histology of the ovary. Gap junctions between the zona pellucida and ovary also failed to form. Induced antibody titre was found to increase the effect of infertility. Furthermore, mice who could not produce antibodies were not rendered infertile by this method.

Unfortunately, viral growth using rMCMV-mZP3 was very poor. One possible explanation is that expression of the foreign antigen led to an increased immune response from the mouse, resulting in poor viral growth. Because viral growth was poor in the saliva, mice did not spread this virus by biting each other (which is how MCMV is usually spread). Therefore, this method of immunocontraception was unable to provide widespread contraception of mice.

Another thing that was learned from this study is that the viral vector used is important. Mice who have innate resistance to the viral vector have a reduced response. The Cmv1 locus appears to be important in MCMV resistance. BALB/c mice have a more profound response to rMCMV-mZP3 as compared to C57BL/6, which have a more "resistant" allele on the Cmv1 locus. However, when C57BL/6 mice were given the G4 (Geraldton) strain of MCMV, which they were not resistant to, they had a strong response to the immunocontraceptive therapy.

Only one more lecture to cover before the test tomorrow!