A CRISPR-Cas9 injection into a cell under the microscope

Precision Medicine and Genomic Resources

Critical goals of NHP research include developing genetically precise NHP models for studying the pathogenesis of human genetic diseases, identifying NHP orthologs of human disease-associated alleles, and developing therapeutic strategies for genetic diseases, including regenerative medicine approaches using iPSCs and targeted gene therapies.

This Resource Unit aims to integrate embryonic and cellular genome editing to develop NHP models, advance in vivo cellular targeting and transplantation, and discover “phenotype to genotype” associations by whole genome sequencing analyses. The unique resources developed by the Unit will be available to WNPRC investigators and the broad scientific community.

RESEARCH GOALS

  • Optimize delivery of CRISPR-Cas9 genome editing constructs to NHP cells and embryos.
  • Advance precision MHC-defined NHP models for gene and stem cell-based therapies for AIDS.
  • Expand and formalize Genomics Resources to support investigators in employing genomic data from nonhuman primate models in their research.

Optimizing Cell and Embryo Genome Editing Platforms

CONTACTS

Current Research

  • Targeting CYP19A1 in rhesus macaque cells and embryos to create an aromatase deficiency model. Aromatase deficiency leads to hyperandrogenism, a feature of polycystic ovarian syndrome in humans.
  • Correction of a naturally-occurring mutation, MAPT R406W, in rhesus macaque cells to develop cell-based gene therapies for frontotemporal dementia.

Activities Supported

  • Provide nonhuman primate fibroblasts, embryonic, induced pluripotent, and trophoblast stem cells (ESC, iPSC, and TSC) for testing CRISPR/Cas9 targeting vectors.
  • Guide CRISPR/Cas9 construct design and optimization of in vitro cell cultures
  • Collect oocytes and sperm for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), providing technical expertise in microinjection and embryo culture.
  • Transfer of in vitro produced embryos to surrogate females and monitor the progress of the pregnancies. Sampling and testing of the offspring for the genetic modification of choice.
  • Tailor project-specific neonatal husbandry to the predicted phenotype of the edited offspring.
  • Develop sperm and embryo cryopreservation for experimental and germplasm banking.

Advancing Precision Therapies

CONTACTS

Current Research

  • Developing MHC-defined model off-the-shelf CAR-T and CAR-NK therapies for HIV infection.
  • Developing non-genotoxic conditioning regimens for transplantation of gene-edited hematopoietic stem cells (HSCs).

Activities Supported

  • Generate and characterize immune effector cells derived from NHP-iPSCs
  • In vivo administration of iPSC-derived hematopoietic progenitors and immune cells
  • Genetic editing of HSCs and iPSCs using CRISPR/Cas9
  • Isolate HSCs from bone marrow and peripheral blood
  • Transplant HSCs and develop novel conditioning regimens
  • Evaluate preclinical iPSC-derived vascular cells

Genomic Discovery

CONTACTS

Current Research

  • Accelerating NHP model development through discovery of naturally-occurring mutations.
  • Analysis of existing and future genomic data to support phenotype-to-genotype and genotype-to-phenotype studies.
  • Comprehensive genomic analysis of CRISPR/Cas9 edited NHP cells and embryos, complementing genome editing and precision therapies research.

Activities Supported

  • Support and assist WNPRC researchers and external investigators in the analysis of genomic data.
  • Provision of sequencing and bioinformatic support for WNPRC researchers and external investigators seeking to perform whole genome or exome sequencing analysis.
  • A goal of Genomic Discovery is to expand and formalize Genomic Resources for investigators to utilize a rapidly increasing NHP genomic database. The PMGR unit offers fee-for-service sequencing and bioinformatic services, in addition to consultation on genomic studies. Investigators seeking genomic support should contact Jeffrey Rogers, Ph.D. Investigators interested specifically in MHC or other immune loci genotyping should contact the WNPRC Genomics Services unit.   

SHARED RESOURCES

A goal of the PMGR unit is to share NHP cell and genomic resources with the broad scientific community.

The following cell resources are available for distribution:

Species PMGR  Unit SCR Unit
mutated wild-type (control) wild-type (control)
Cynomolgus  CCR5-knockout iPSC fibroblasts

trophoblast stem cells

fibroblasts

iPSCs

Rhesus fibroblasts

trophoblast stem cells

fibroblasts

ESCs, iPSCs

Marmoset LRRK2 G2019S ESC

LRRK2 G2019S iPSC

fibroblasts ESCs, iPSCs

Highlighted Publications

Schmidt JK, Block LN, Jones KJ, Hinkle HM, Mean KD, Bowman BD, Makulec AT, Golos TG. Atypical initial cleavage patterns minimally impact rhesus macaque in vitro embryo morphokinetics and embryo outgrowth development. Biol Reprod. 2023 Sep 9:ioad117.

Neuman SS, Metzger JM, Bondarenko V, Wang Y, Felton J, Levine JE, Saha K, Gong S, Emborg ME. Striatonigral distribution of a fluorescent reporter following intracerebral delivery of genome editors. Front Bioeng Biotechnol. 2023 Jul 26;11:1237613.

Fiziev PP, McRae J, Ulirsch JC, Dron JS, Hamp T, Yang Y, Wainschtein P, Ni Z, Schraiber JG, Gao H, Cable D, Field Y, Aguet F, Fasnacht M, Metwally A, Rogers J, Marques-Bonet T, Rehm HL, O’Donnell-Luria A, Khera AV, Farh KK. Rare penetrant mutations confer severe risk of common diseases. Science. 2023 Jun 2;380(6648):eabo1131.

Gao H,…Rogers J, et al. The landscape of tolerated genetic variation in humans and primates. Science. 2023 Jun 2;380(6648):eabn8153.

Kuderna LFK,…Rogers J, et al. A global catalog of whole-genome diversity from 233 primate species. Science. 2023, June 2, 380(6648):906-913.

Marmoset LRRK2 G2019S ESC LRRK2 G2019S iPSC fibroblasts ESC, iPSC Gambardella JC, Schoephoerster W, Bondarenko V, Yandell BS, Emborg ME. Expression of tau and phosphorylated tau in the brain of normal and hemiparkinsonian rhesus macaques. J Comp Neurol. 2023 Apr 26.

Jung HS, Suknuntha K, Kim YH, Liu P, Dettle ST, Sedzro DM, Smith PR, Thomson JA, Ong IM, Slukvin II. SOX18-enforced expression diverts hemogenic endothelium-derived progenitors from T towards NK lymphoid pathways. iScience. 2023 Apr 8;26(5):106621.

Kabakov SA, Crary E, Menna V, Razo ER, Eickhoff JC, Dulaney NR, Drew JR Jr, Bach KM, Poole AM, Stumpf M, Mitzey AM, Malicki KB, Schotzko ML, Pickett KA, Schultz-Darken NJ, Emborg ME, O’Connor DH, Golos TG, Mohr EL, Ausderau KK. Quantification of early gait development: Expanding the application of Catwalk technology to an infant rhesus macaque model. J Neurosci Methods. 2023 Mar 15;388:109811.

Metzger JM, Wang Y, Neuman SS, Snow KJ, Murray SA, Lutz CM, Bondarenko V, Felton J, Gimse K, Xie R, Li D, Zhao Y, Flowers MT, Simmons HA, Roy S, Saha K, Levine JE, Emborg ME, Gong S. Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins. Biomaterials. 2023 Feb;293:121959.

Olsen ME, Brodsky EK, Oler JA, Riedel MK, Mueller SAL, Vermilyea SC, Metzger JM, Tao Y, Brunner KG, Ahmed AS, Zhang SC, Emborg ME, Kalin NH, Block WF. Real-time trajectory guide tracking for intraoperative MRI-guided neurosurgery. Magn Reson Med. 2023 Feb;89(2):710-720.

Vallender EJ, Hotchkiss CE, Lewis AD, Rogers J, Stern JA, Peterson SM, Ferguson B, Sayers K. Nonhuman primate genetic models for the study of rare diseases. Orphanet J Rare Dis. 2023 Jan 31;18(1):20.

Ausderau KK, Colman RJ, Kabakov S, Schultz-Darken N, Emborg ME. Evaluating depression- and anxiety-like behaviors in non-human primates. Front Behav Neurosci. 2023 Jan 19;16:1006065.

Schmidt JK, Kim YH, Strelchenko N, Gierczic SR, Pavelec D, Golos TG, Slukvin II. Whole genome sequencing of CCR5 CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits. Front Genome Ed. 2023 Jan 12;4:1031275.

Zhang J, Webster S, Duffin B, Bernstein MN, Steill J, Swanson S, Forsberg MH, Bolin J, Brown ME, Majumder A, Capitini CM, Stewart R, Thomson JA, Slukvin II. Generation of anti-GD2 CAR macrophages from human pluripotent stem cells for cancer immunotherapies. Stem Cell Reports. 2022 Dec 28:S2213-6711(22)00604-X.

Emborg ME. Reframing the Perception of Outliers and Negative Data in Translational Research. Brain Res Bull. 2022 Dec 1:S0361-9230(22)00328-8.

D’Souza SS, Kumar A, Maufort J, Weinfurter JT, Raymond M, Strelchenko N, Perrin E, Coonen J, Mejia A, Simmons H, Torbett BE, Reynolds M, Thomson JA, Slukvin I. Assessment of Safety and Immunogenicity of MHC homozygous iPSC-derived CD34+ Hematopoietic Progenitors in a NHP Model. Blood Adv. 2022 Sep 27;6(18):5267-5278.

Schmidt JK, Reynolds MR, Golos TG, Slukvin II. CRISPR/Cas9 genome editing to create nonhuman primate models for studying stem cell therapies for HIV infection. Retrovirology 2022, 19:17. 2022 Aug 10.

Weinfurter JT, D’Souza SS, Matschke LM, Bennett S, Kelnhofer-Millevolte LE, Suknuntha K, Kumar A, Coonen J, Capitini CM, Hematti P, Golos TG, Slukvin II, Reynolds MR. Allogeneic MHC-matched T-cell receptor α/β-depleted bone marrow transplants in SHIV-infected, ART-suppressed Mauritian cynomolgus macaques. Sci Rep. 2022 Jul 19;12(1):12345.

Zinnen AD, Vichich J, Metzger JM, Gambardella JC, Bondarenko V, Simmons HA, Emborg ME. Alpha-synuclein and tau are abundantly expressed in the ENS of the human appendix and monkey cecum. PLoS One. 2022 June 17:e0269190.

Uenishi GI, Jung HS, Slukvin II. Assessment of Endothelial-to-Hematopoietic Transition of Individual Hemogenic Endothelium and Bulk Populations in Defined Conditions. Methods Mol Biol. 2022;2429:103-124.

D’Souza SS, Kumar A, Maufort J, Weinfurter JT, Raymond M, Strelchenko N, Perrin E, Coonen J, Mejia A, Simmons H, Torbett BE, Reynolds M, Thomson JA, Slukvin I. Assessment of Safety and Immunogenicity of MHC homozygous iPSC-derived CD34+ Hematopoietic Progenitors in a NHP Model. Blood Adv. 2022 Apr 11:bloodadvances.2022006984.

D’Souza SS, Kumar A, Weinfurter J, Park MA, Maufort J, Tao L, Kang H, Dettle ST, Golos T, Thomson JA, Reynolds MR, Slukvin I. Generation of SIV-resistant T cells and macrophages from nonhuman primate induced pluripotent stem cells with edited CCR5 locus. Stem Cell Reports. 2022 Mar 17:S2213-6711(22)00136-9.

Schmidt JK, Jones KM, Van Vleck T, Emborg ME. Modeling genetic diseases in nonhuman primates through embryonic and germline modification: Considerations and challenges. Sci Transl Med. 2022 Mar 2;14(634):eabf4879.