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Page 6 of 13 Li et al. Ageing Neur Dis 2022;2:13 https://dx.doi.org/10.20517/and.2022.13

It is apparent that the phenotypes of transgenic HD pig models are dependent on the expression levels of
transgenic N-terminal mutant HTT. It is important to create a pig model that expresses full-length mutant
HTT at the endogenous level. With the development of CRISPR/Cas9 technology, precise gene editing of
[33]
[32]
various animal species becomes possible , especially for the generation of large animal models . To
overcome the shortcomings of the transgenic pig model of HD, Yan et al. first used CRISPR/Cas9 to insert a
large CAG repeat (150 CAGs) into the pig HTT locus in fibroblast cells, and then used SCNT to generate a
HD knock-in pig model . The brains of this pig model showed severe and preferential neurodegeneration
[50]
in the medium spiny neurons in the striatum, an important pathological feature in HD patients. More
importantly, the HD pig models displayed dance-like symptoms and breathing difficulties, which were
similar to the symptoms in HD patients. Further, the pathogenic and neurologic features of HD pigs can be
stably passed to offspring, enabling the establishment of a large animal model of HD for mechanistic study
and drug screening.

Pig models of Alzheimer’s disease
The incidence rate of Alzheimer’s disease (AD) is increasing year by year with aging. Its early neurological
symptoms are mainly memory loss and behavioral changes, and, in the late stage, the patients will have
cognitive impairment, which severely affects daily life . AD is usually divided into familial AD (FAD) and
[51]
sporadic AD (SAD) according to different pathologies. Only about 5% of AD cases are FAD and are caused
by mutations in β-amyloid precursor protein (APP), presenilin 1 (PS1), and/or presenilin 2 (PS2). Nearly
95% of patients with AD are classified as SAD, which is caused by a combination of genetic factors and
environmental risk factors without documented familial history of AD . The deposition of β-amyloid (Aβ)
[52]
and hyperphosphorylation of Tau are the major pathological hallmarks, with other pathophysiologic
changes including neuroinflammation, oxidative stress, and abnormal lipid metabolism. In addition to Aβ
and Tau, apolipoprotein E4 (APOE4) and coulomb-receptor expressed on myeloid cells 2 (TREM2) are
considered to be the risk factors . Various mouse models of AD have been developed to mimic the
[53]
symptoms of AD. However, due to the complexity of the neuropathology spectrum of AD, none of the
available mouse models truly recapitulate the full spectrum of AD neuropathology, which includes Aβ
deposition, synapse loss, inflammation, tau hyperphosphorylation, and neurofibrillary tangle formation .
[54]
To model the characteristics of AD in more human-like species, researchers injected Aβ oligomers into the
lateral ventricle of macaques, which diffused into the brain and accumulated in several regions associated
with memory and cognitive functions. They found that oligomer injections induced AD-like pathology with
[55]
neurofibrillary tangle formation in the macaque brain, which was not found in small animal models .
Other researchers also used viral delivery of human 4R-tau to generate a tau-based rhesus monkey model of
[56]
Alzheimer’s disease . However, due to the long reproductive cycle of monkeys and immature cloning
technology, it was difficult to obtain a large group of monkey models of AD through transgenic methods.
Therefore, the establishment of transgenic pig models of AD is needed.

In 2009, Kragh et al. tried to develop a pig model of Alzheimer’s disease by expressing AD-causing
sw
dominant mutation APP . The transgene consisted of the cDNA of the neuronal variant of the human APP
gene with the Swedish mutation. However, no disease phenotype was reported, although it was predicted
that accumulation of the Aβ peptide in the brain might develop at the age of 1-2 years . The same group
[57]
also generated a transgenic miniature pig model expressing a cDNA of the AD-causing gene PSEN1M146I
driven by an enhanced human UbiC promoter. However, no phenotypic data have been published yet [46,58] .
To induce the neuropathology of the increased intraneuronal Aβ plaque formation, this group combined
the mutation of PSEN1 and APP together to generate double transgenic Göttingen minipigs that carry one
copy of a human PSEN1 cDNA with the Met146Ile (PSEN1M146I) mutation and three copies of a human A
βPP695 cDNA with the Lys670Asn/Met671Leu (AβPPsw) double mutations. Their strategy successfully
generated a pig model with an intraneuronal accumulation of Aβ42 in the brain between the age of 10 and