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Non‐human primates in the research of neurological diseases
Yingjun Liu07.03.2017
Interdisciplinary Technical Journal Club: special series on Laboratory Animal Science
Neurological disorders: challenges of drug disovery
• CNS drug discovery and development has been problematic, particularly in the area of acute and chronic neurodegenerative disorders.
• These difficulties have resulted in significant cutbacks in CNS drug programs.
• Drug targets must be better understood in terms of their role in normal CNS function and in disease.
• Animal models of disease and hypothetical drug regimens must better reflect the clinical condition.
Valentin K. Gribkoff and Leonard K. Kaczmarek, Neuropharmacology, 2016US Burden of Disease Collaborators, JAMA. 2013
DALYS (Disability‐adjusted life‐years): a summary metric of population health. DALYs measure the state of a population’s health compared to a normative goal. The goal is for individuals to live the standard life expectancy in full health. DALYs are the sum of 2 components: years of life lost (YLLs) and years lived with disability (YLDs).
Genomic responses in mouse models poorly mimic human conditions
Junhee Seok, H. Shaw Warren, Alex G. Cuenca et al., PNAS, 2013Terry C. Burns et al., European Journal of Pharmacology, 2015
Why do we use NHPs in research?
Asao Fujiyama, Hidemi Watanabe, Masahira Hattori and YoshiyukiSakaki et al., Science, 2002Roy J. Britten, PNAS, 2002
DNA sequence similarities (Human v.s Chimpanzee): around 98%. 95% (When considering the indels)
Clark Spencer Larsen, Our Origins: Discovering Physical Anthropology, 2nd edition, 2011.
From Central and South‐AmericaSmallest monkeys in the worldAverage hieghts, 188 mm. Average weights, 256 g. (Rowe 1996).Marmoset
What kind of NHPs do we use in research?
From East and South‐Asia, the Middle East and AfricaAverage hieghts, 531.8 mm Average weights, 7.7 kg (Fooden 2000; Singh & Sinha 2004)
Macaque
From AfricaThe closest human relativesAverage hieghts, 816 mm Average weights, 50 kg (Rowe 1996)Chimpanzee
https://www.mrc.ac.uk/documents/pdf/the‐use‐of‐non‐human‐primates‐in‐research/http://ec.europa.eu/health/ph_risk/committees/04_scher/docs/scher_o_110.pdfhttp://eur‐lex.europa.eu/LexUriServ/LexUriServ.do?uri=COM:2013:0859:FIN:EN:PDFhttp://www.navs.org/what‐we‐do/keep‐you‐informed/science‐corner/animals‐used‐in‐research/nonhuman‐primates‐in‐research/#.WKrRTG8rK70
How many NHPs do we use in research?
NHPs in biomedical research
Advantages
• Neuroscience• Toxicology• Infections diseases• Immunology• Reproduction
Limitations
• Expensive to maintain• Time‐consuming breeding• Difficulty in genetical
manipulations• Increasing Ethical concrens• NPHs are not humans
NHPs naturally display Alzheimer's disease‐like lesions
Eric Heuer, Rebecca F. Rosen, Amarallys Cintron, Lary C. Walker, Curr Pharm Des, 2012
Human AD Aged monkey
Human AD Aged monkey Human AD Aged monkey
Eric Heuer, Rebecca F. Rosen, Amarallys Cintron, Lary C. Walker, Curr Pharm Des, 2012Jean‐Luc Picq and Marc Dhenain et al., Neurobiol Aging, 2012
r = 0.60
r = 0.61
Contributions of NHPs in biomedical research: Parkinson’s diseases
Davis GC and Kopin IJ et al., Psychiatry Res., 1979R. STANLEY BURNS AND IRWIN J. KOPIN et al., Proc. Natl. Acad. Sci. 1983
Meperidine
Normal MPTP
MPTP
MPP+
H Bergman, T Wichmann, MR DeLong, Science, 1990Adriana Galvan, Thomas Wichmann, Clinical Neurophysiology, 2008Mahlon R. DeLong, Alim‐Louis Benabid, JAMA., 2014
+IBO
Con
DBS • https://www.youtube.c
om/watch?v=wZZ4Vf3HinA
• https://www.youtube.com/watch?v=0n12yPOUumg
Genetical modifications in NHPs: the obstacles
• Obtaining large numbers of oocytes from NPHs is limited by ethical and economic constraints
• Traditional pronuclear microinjection approach is not efficient in NHPs
• NHP ES cells are very difficult to maintain and more susceptible to various stresses
• Difficulty in SCNT for NPHs
Transgenic Knock-out and knock in Cloning (SCNT)
Genetical modifications in NHPs: the first GM monkey in the world‐ANDi
A. W. S. Chan and G. Schatten et al., Science, 2001* Twin pregnancy.
Genetical modifications in NHPs: the first GM monkey in the world‐ANDi
• ANDi was the first transgenic monkey, born in 2000.• “ANDi” stands for “inserted DNA” spelled backwards.• An engineered virus was used to insert the harmless gene for green
fluorescence protein (GFP) into ANDi’s rhesus genome.• ANDi proves that transgenic primates can be created, and can express a
foreign gene delivered into their genome.
ANDi An male stillborn
Genetical modifications in NHPs: Towards a transgenic model of Huntington’s disease
Shang‐Hsun Yang and Anthony W. S. Chan et al., Nature, 2008Anthony W. S. Chan and Jocelyne Bachevalier et al., Plos one, 2015
Genetical modifications in NHPs: Germ‐line transmission
• Sexual maturity at 12–18 months• females have 40–80 offspring during their life
From Central and South‐AmericaSmallest monkeys in the worldAverage hieghts, 188 mm. Average weights, 256 g. (Rowe 1996).Marmoset
From East and South‐Asia, the Middle East and AfricaAverage hieghts, 531.8 mm Average weights, 7.7 kg (Fooden 2000; Singh & Sinha 2004)
• Sexual maturity about 3 years• females have around 10 offspring during their life
• Marmoset embryos at the pronuclear‐to‐morula stage
• Lentiviral injection into perivitelline space
Approaches
Macaque
Pronuclear Morula Blastocyst
Kathy K. Niakan and Renee A. Reijo Pera et al., 2012, Development
4‐cell stage 8‐cell stage
Zona pellucida
Perivitelline space
0.25 M sucrose in PB1 medium (0.25 M sucrose medium), which made the perivitelline space expand 1.2–7.5‐fold
Erika Sasaki and Hideyuki Okano et al., Nature, 2009
Yuyu Niu, Yang Yue and Weizhi Ji et al., PNAS, 2010Yuyu Niu, Weizhi Ji and Xiao‐Jiang Li et al., Hum Mol Genet, 2015
• Low efficiency
• Based on virus system
• Difficulty in phenotype characterizytion
• Difficulty in targeted genetic
manipulations (like knockout or
matugenesis)
Limitations for all these early attempts forthe generation of GM NPHs
α-syn (A53T) tg Monkey
MECP2 Duplication syndrome
• Hypotonia (floppiness or low muscle tone).• Absent or little speech.• Severe learning difficulty.• Progressive neurological conditions such as seizures• Autistic like behaviors such as hand flapping and rocking• Ataxia (balance and control issues)
MECP2 mutation and Rett Syndrome
Thierry Bienvenu & Jamel Chelly, Nature Reviews Genetics, 2006Zhen Liu, Xiao Li, Qiang Sun & Zilong Qiu et al., Nature, 2016
• Loss of normal movement and coordination. • Loss of communication abilities• Abnormal hand movements. • Agitation and irritability. • Cognitive disabilities.
Zhen Liu, Xiao Li, Qiang Sun & Zilong Qiu et al., Nature, 2016
Zhen Liu and Qiang Sun et al., Cell Research, 2016Zhen Liu, Xiao Li, Qiang Sun & Zilong Qiu et al., Nature, 2016
Generation of macaques with sperm derived from juvenile monkey testicular xenografts
Shorten the generation of cynomolgus monkey offspring by 1‐2 years
TALEN‐mediated genome editing in the development of GM NHPs
TALEN: Transcription activator-like (TAL) effectors nuclease
Tomas Cermak, Adam J. Bogdanove and Daniel F. Voytas et al., Nucleic Acids Res. 2011Hailiang Liu, Siguang Li, Yi Eve Sun and Weizhi Ji et al., Cell stem cell, 2014
• Human and primate MECP2 genes are targeted effectively by three TALEN pairs
• Plasmid‐based targeting in primate zygotes leads to mutagenesis
• Extensive analysis detects no evidence of off‐target activity
• Male fetuses show midgestationlethality but a female was live born
Hailiang Liu, Siguang Li, Yi Eve Sun and Weizhi Ji et al., Cell stem cell, 2014
CRISPR/Cas9‐Mediated Gene Targeting in the development of GM NHPs
http://www.origene.com/CRISPR‐CAS9/Yuyu Niu, Xingxu Huang, Weizhi Ji and Jiahao Sha et al., Cell, 2014
• CRISPR/Cas9‐mediated simultaneous targeting of multiple genes in monkey embryos• Demonstrates an efficient and reliable approach for genome modified monkey generation• Achieves Ppar‐g and Rag1 double mutation in monkeys in one step
Twenty nanogram/μl Cas9 mRNA and 25 nanogram/μl of mixtures containing equal amount of each 5 sgRNAs were pooled and microinjected into 22 one-cell fertilized eggs of cynomolgus monkeys.
Yuyu Niu, Xingxu Huang, Weizhi Ji and Jiahao Sha et al., Cell, 2014
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