NANOS knock-out that ablates germline cells
Inventors
Oatley, Jon Michael • Whitelaw, Christopher Bruce Alexander • LILLICO, Simon Geoffrey • Telugu, Bhanu Prakash
Assignees
University of Edinburgh • University of Maryland Baltimore • Washington State University WSU
Publication Number
US-12102069-B2
Publication Date
2024-10-01
Expiration Date
2035-07-14
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Abstract
The present invention provides livestock animals and methods to create recipient animals for spermatogonial stem cell transplantation through modulation of the NANOS gene. In one embodiment genome editing issued to create animals with insertions or deletions (indels) that inactivate or otherwise modulate NANOS gene activity so that resulting males lack functional germ cells yet retain functional somatic cells, and females are fertile. These males can then be transplanted with donor spermatogonial stem cells and used for breeding.
Core Innovation
The invention provides genetically edited livestock animals and methods that enable the creation of recipient animals for spermatogonial stem cell (SSC) transplantation by modulating the expression of the NANOS gene. Specifically, genome editing methods such as CRISPR/Cas9, TALENs, Zinc Finger Nucleases, or recombinase fusion proteins are used to introduce insertions or deletions (indels) that inactivate or modulate NANOS gene activity. As a result, male animals lack functional germline cells but retain functional somatic cells such as Sertoli cells, while females remain fertile.
The problem addressed by this invention is that current methods for preparing recipient animals for SSC transplantation, such as treating with chemotoxic drugs or irradiation, do not completely eliminate endogenous germ cells and may harm somatic cell health, leading to reduced transplantation efficiency and poor colonization of donor SSCs. In pigs and large domestic animals, these approaches are impractical due to high toxicity, difficulty in dose control, and biohazard risks. There is a significant need for recipient animals that are genetically deficient in germline cells but retain intact support cell populations, enabling unimpeded engraftment of donor SSCs.
The innovation centers on livestock animals, especially pigs, that are edited to possess inactivated NANOS2 genes (e.g., homozygous knockouts or bi-allelic null mutations), resulting in males that are sterile with no endogenous germ cells and normal somatic support cells, and females that remain fertile. These animals serve as ideal recipients for SSCs harvested and cultured from desired donor males. The donor SSCs are transplanted into the testes of NANOS2 knockout males, allowing the regeneration and persistence of donor-derived spermatogenesis for breeding, including both artificial insemination and natural mating.
Claims Coverage
The patent claims cover several inventive features related to the genetic modification of the NANOS2 gene in male porcine animals to produce germline-ablated, somatically intact recipients for donor spermatogonial stem cell transplantation and subsequent use in breeding.
Method for producing NANOS2 knockout male porcine recipients supporting SSC transplantation
A method is provided that involves modifying NANOS2 gene expression in a male porcine animal by creating insertion or deletion (indel) mutations leading to a homozygous or bi-allelic NANOS2 gene knockout, resulting in reduced or eliminated NANOS2 protein function. The modified animal lacks germline cells but retains Sertoli cell function. The modified NANOS2 gene comprises specific listed sequences (SEQ ID NOs: 27, 28, 30, 31, 33, 34, 36, 37, 38, 40, 41, 42, 45, 46, 48, 49, 51, 56, 57, 164, 165, 166, 167, 168, 169, 172, 176, 177, 180, 181, 184, 185, 186, 190, 192, 193, 195, 199, 201, 203, or 204). The method includes transplanting donor spermatogonial stem cells (SSCs) into the male porcine animal so that spermatogenic colonies are generated, and subsequently introducing donor-derived sperm from this male into a female porcine animal to achieve pregnancy. The invention specifies that the male recipient may have no exogenously introduced sequence, and that the introducing step may be by artificial insemination or natural mating.
Use of site-specific gene editing reagents for NANOS2 gene modification
The inventive method includes use of guide RNAs targeting the NANOS2 gene and polypeptides capable of inducing cleavage or integration at the target site. Specifically, site-specific genome editing tools such as RNA-guided CRISPR/Cas9, TALENs, zinc finger nucleases, and recombinase fusion proteins comprising a NANOS2 site-specific DNA binding domain and a nuclease cleavage domain are claimed for generating the required knockout mutations.
Donor spermatogonial stem cell (SSC) collection, culture, and transplantation protocols
The patent further claims steps where donor SSCs are collected from a desired male donor, optionally proliferated in vitro, and then transplanted into the NANOS2 knockout male by injection into the rete testis. The method ensures generation of donor-derived spermatogenic colonies in the recipient animal.
Breeding using donor-derived sperm via natural mating or artificial insemination
The inventive features include introducing donor-derived sperm from the NANOS2 knockout male, following successful colonization by donor SSCs, into a female porcine animal either via artificial insemination or natural mating so that the female becomes pregnant.
These inventive features define the scope of the patent as methods and compositions for generating genetically edited male porcine recipients that lack germline cells but maintain functional Sertoli cells through specific NANOS2 gene knockouts; transplantation of donor SSCs into these recipients; and use of resultant donor-derived sperm in breeding processes.
Stated Advantages
The method produces male recipients lacking endogenous germ cells but retaining intact somatic support cells, providing an ideal environment for donor SSC engraftment and regeneration of spermatogenesis.
Unlike chemotoxic or irradiation approaches, the invention avoids toxicity, biohazard risks, and damage to support cells, enabling efficient, healthy donor SSC colonization and exclusive donor-derived sperm production.
The breeding method expands the output and availability of gametes from desirable sires, preserving and propagating germline and genetic merit without limitations of traditional artificial insemination.
Documented Applications
Preparation of male porcine animals as recipients for spermatogonial stem cell transplantation by editing the NANOS2 gene, enabling germline ablation and use in breeding programs.
Transplantation of donor spermatogonial stem cells into NANOS2 knockout recipients and subsequent use of resultant donor-derived sperm for artificial insemination or natural mating to produce offspring.
Preservation and propagation of the genetic merit or specific traits of desired sires through expansion of donor SSCs, their transplantation, and use of donor-derived sperm in livestock production.
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