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Li et al. Ageing Neur Dis 2022;2:12 https://dx.doi.org/10.20517/and.2022.14 Page 7 of 13

transmissible with a stable expression level [127,128] . Importantly, the offspring of the founder animal displayed
[129]
CAG repeat instability, a critical feature of HD . In subsequent longitudinal investigations, researchers
further revealed that this model could resemble progressive striatal and hippocampal morphometric
changes as well as motor and cognition impairment, similar to clinical observation [130,131] .

Recently, several transgenic swine HD models were also generated [132-134] ; these models also showed more
obvious neurodegeneration and motor disorders than that of mice with the same length of CAG repeats.
Through CRISPR/Cas9-mediated knock-in (KI) on pig fibroblast cells and somatic cell nuclear transfer
[135]
technique, a HD KI pig model was successfully created . This model expresses 150 CAG repeats under the
endogenous HTT promoter, leading to selective neurodegeneration as well as movement disorders,
effectively mimicking typical HD pathology and clinic features. Importantly, similar to the transgenic HD
[135]
monkey, the HD KI pig is also inheritable , making it possible to generate a large number of HD pigs in
the future based on the potent reproductive capability of swine. Although the HD KI pig seems to be an
ideal model, the macaque monkey model is more suitable for investigating emotional and psychiatric
activity. Thus, combining HD pig and monkey models will bring us deeper insight into HD pathogenesis as
well as advances in HD therapy.

CHALLENGES AND SOLUTIONS
With the development of gene editing technology, especially CRISPR/Cas9, several NHP models of NDs
have been established and offer new insights into pathogenesis. For example, aging has been confirmed to
be critical for age-dependent neurodegeneration, as expressing the disease proteins (Amyloid-β, Tau, and
α-Synuclein) in the brains of old monkeys can faithfully recapitulate neuropathology [82,85,99] . Investigations on
NHP models of NDs also revealed that species-dependent factors are critically involved in important
pathological events. Depletion of PINK1 can lead to severe neuronal loss in the monkey brain but not in the
mouse brain, which is largely due to abundant expression of PINK1 in the primate brain and undetectable
level of PINK1 in the rodent brain [101,103] . The lack of cytoplasmic distribution of mutant TDP-43 in most
mouse models is perhaps due to the absence of caspase-4, a primate-specific enzyme that can cleave TDP-43
[119]
to cause truncated TDP-43 to move from the nucleus to the cytoplasm . With more in-depth studies and
the establishment of additional non-human primate models of NDs, greater advances are expected, which
will bring new insights into disease pathogenesis.

Despite the great progress that has been made, NHP models of NDs have not been used at a large scale. One
key hurdle is that most models are hard to scale up for widespread application. Due to relatively low
reproductive capability, a longer time for sexual maturity, and the absence of germline integrating ESC, it is
challenging to generate NHP models by following rodent protocols to knock-in or knock-out the
[136]
endogenous genes in one-cell stage embryos . Although cloning of the macaque monkey , especially by
[7]
somatic cell nuclear transfer [137,138] , has been demonstrated to be feasible, the efficiency is suboptimal and
remains to be improved. Since many NDs are caused by point mutation, newly emerging base editing and
prime editing tools [139,140] will facilitate the generation of better animal models that can precisely mimic
human genetic mutations. Recent research indicates that appropriate small molecules are able to direct
[141]
human ESC to the early stage blastomere state , suggesting the opportunity to implement a germline
integration strategy in non-human primates [Figure 1].

Aging is the biggest risk factor for ND incidence. When investigating animal models generated from
germline genetic manipulations, the animals are likely to show phenotypes and neuropathology when they
become old. In this regard, any treatments that can promote the aging process should presumably facilitate
the development of disease phenotypes. Considering the high cost for maintaining non-human primates