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Developing a Non-human Primate Model for the Evaluation of Novel Stem cell-based HIV Therapeutics

Review ofGenome editing of CCR5 by CRISPR-Cas9 in Mauritian cynomolgus macaque embryosfrom Scientific Reports by Stuart P. Atkinson

Various studies have demonstrated that a homozygous 32 base-pair deletion in the chemokine (C–C) motif receptor 5 (CCR5) gene leads to a truncation of the CCR5 protein, the loss of cell surface expression 6, and resistance to human immunodeficiency virus-1 (HIV-1) infection in humans [1-3]. The exploration of CCR5 as the basis for potential HIV protective regimens or treatments will require further study in relevant non-human primate models, such as simian immunodeficiency virus (SIV) infection in Mauritian cynomolgus macaques [4].

Previous research led by Thaddeus G. Golos and Igor I. Slukvin (University of Wisconsin-Madison, Madison, WI, USA) described how the CRISPR/Cas9-mediated disruption of CCR5 in human induced pluripotent stem cells permitted the generation of macrophages that displayed resistance to HIV challenge [5]. In their most recent study in this area [6], the team now reports on the development of a CCR5-edited Mauritian cynomolgus macaque model via the CRISPR/Cas9-mediated gene-editing of embryos.

Schmidt et al. modified an established protocol for the production of in-vitro fertilization embryos from Chinese cynomolgus macaques [7, 8] to generate Mauritian cynomolgus macaque embryos. Following an optimized ovarian stimulation protocol, the collection of mature oocytes by laparoscopic follicular aspiration, and an in vitro maturation step, the team fertilized mature oocytes by intracytoplasmic sperm injection. Approximately six hours later, the authors then microinjected the Cas9 protein and dual-guide RNAs designed to target sequences within exon 2, including a 24 base-pair deletion region that prevented functional CCR5 expression in non-human primates [9]. Overall, the microinjected embryos displayed a significant reduction in cleavage rates compared to control embryos, with time-lapse embryo imaging displaying delays in developmental timing. Over 50% of assayed embryos contained the predicted deletion in CCR5, and over 35% displayed biallelic deletions; however, no pregnancies were obtained following the careful selection of embryo recipients and using an embryo transfer protocol previously employed successfully with rhesus macaque embryos [10].

While this new study has described the first steps towards creating an SIV resistance model that will aid our understanding of HIV progression and resistance and allow for the evaluation of advanced stem cell-based therapeutics, the authors point to the required next steps in their research. These include the optimization of in vitro embryo culture conditions, the improvement of editing efficiency without negatively impacting embryonic development, and the evaluation of possible off-target editing.

For more on the creation of new models that will aid our understanding of HIV development and foster the generation of effective new therapies for this devastating disease, stay tuned to the Stem Cells Portal!

References

  1. Liu R, Paxton WA, Choe S, et al., Homozygous Defect in HIV-1 Coreceptor Accounts for Resistance of Some Multiply-Exposed Individuals to HIV-1 Infection. Cell 1996;86:367-377.
  2. Samson M, Libert F, Doranz BJ, et al., Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 1996;382:722-725.
  3. Dean M, Carrington M, Winkler C, et al., Genetic Restriction of HIV-1 Infection and Progression to AIDS by a Deletion Allele of the CKR5 Structural Gene. Science 1996;273:1856.
  4. Liang B, Li H, Li L, et al., Current advances in HIV vaccine preclinical studies using Macaque models. Vaccine 2019;37:3388-3399.
  5. Kang H, Minder P, Park MA, et al., CCR5 Disruption in Induced Pluripotent Stem Cells Using CRISPR/Cas9 Provides Selective Resistance of Immune Cells to CCR5-tropic HIV-1 Virus. Molecular Therapy - Nucleic Acids 2015;4.
  6. Schmidt JK, Strelchenko N, Park MA, et al., Genome editing of CCR5 by CRISPR-Cas9 in Mauritian cynomolgus macaque embryos. Scientific Reports 2020;10:18457.
  7. Curnow E and Hayes E, In Vitro Culture of Embryos from the Cynomolgus Macaque (Macaca fascicularis), in Comparative Embryo Culture: Methods and Protocols, J.R. Herrick, Editor. 2019, Springer New York: New York, NY. p. 321-339.
  8. Ma Y, Li J, Wang G, et al., Efficient production of cynomolgus monkeys with a toolbox of enhanced assisted reproductive technologies. Scientific Reports 2016;6:25888.
  9. Chen Z, Kwon D, Jin Z, et al., Natural Infection of a Homozygous Δ24 CCR5 Red-capped Mangabey with an R2b-Tropic Simian Immunodeficiency Virus. Journal of Experimental Medicine 1998;188:2057-2065.
  10. Curnow EC, Pawitri D, and Hayes ES, Sequential culture medium promotes the in vitro development of Macaca fascicularis embryos to blastocysts. American Journal of Primatology 2002;57:203-12.