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Gene Therapy for Male Infertility

Doungkamchan, Chatchanan/CD (2020) Gene Therapy for Male Infertility. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Single gene defects have been associated with non-obstructive azoospermia patients and confirmed in infertile mouse models. Those defects can impact the function of testicular somatic cells (e.g., Sertoli cells) or germ cells. In an infertile Sertoli cell androgen receptor knockout (SCARKO) mouse model, I used adenovirus for in vivo delivery of an androgen receptor (AR) expression vector into Sertoli cells. I retrieved sperm three months later for intracytoplasmic sperm injection (ICSI). Four babies were born from two surrogate mothers, all of which were fertile and transgene-free. For germline gene therapy, I generated a mouse model (Tex11-D435fs) with an analogous mutation to one of our human patients. I transfected Tex11-D435fs spermatogonial stem cell (SSC) culture with CRISPR/Cas9 and an oligonucleotide template to correct the mutated sequence back to the sequence that is common in the human and mouse genome. Gene-corrected SSCs were transplanted into the testes of infertile recipients and established complete spermatogenesis. Sperm were competent to fertilize eggs using ICSI or IVF and produced healthy offspring. For large deletions where insertion of a transgene cassette is required, I used Sohlh1-KO mice with a three-exon deletion. Since Sohlh1+/- mice are fertile, I hypothesized that introducing one normal allele of Sohlh1 cDNA at the “safe harbor” Rosa26 locus would restore spermatogenesis. Because the segregation outcome from Rosa26Tg-Sohlhl1/WT Sohlh1-/- SSCs, would result in 50% transgene-free sperm, half of offspring will be transgene-free. I generated Rosa26Tg-Sohlhl1/WT Sohlh1-/- SSCs and transplanted them into infertile recipients. Spermatogenesis and fertility assessments are underway. Translation to humans is complicated by the lack of robust human SSC culture methods. In contrast, methods to culture and edit human induced pluripotent stem cells (iPSCs) are well established. I acquired the expertise to produce iPSCs from wild type mouse fibroblasts and differentiated them into primordial germ cell-like cells (PGCLCs) that produced spermatogenesis in recipient males and an embryo from iPSC-derived sperm. I then established iPSC lines from Sohlh1-KO mice and used CRISPR/Cas9 gene editing to introduce the Sohlh1 cDNA into the Rosa26 locus. Studies are planned to differentiate those cells into PGCLCs and transplant. These studies demonstrate the feasibility of using somatic cell and germ cell gene therapy to treat male infertility.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Doungkamchan, Chatchanan/CDchd50@pitt.educhd500000-0001-5438-7421
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairOrwig, KEkeo6@pitt.eduKEO6
Committee MemberHukriede, NAhukriede@pitt.eduHUKRIEDE
Committee MemberRajkovic, Aalr110@pitt.eduALR110
Committee MemberWalker, WHwalkerw@pitt.eduWALKERW
Committee MemberHomanics, Gregg E.HomanicsGE@anes.upmc.edu
Date: 15 June 2020
Date Type: Publication
Defense Date: 26 March 2020
Approval Date: 15 June 2020
Submission Date: 27 May 2020
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 203
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Genetics and Developmental Biology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: male infertility azoospermia gene therapy CRISPR Cas9
Date Deposited: 16 Jun 2020 02:09
Last Modified: 16 Jun 2020 02:09
URI: http://d-scholarship.pitt.edu/id/eprint/39123

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