Breakthrough | Li Xiaojiang/Lai Liangxue team uses AAV gene therapy to treat Huntington's disease pigs
Time：2025-04-08 11:07:51
Huntington's disease
Huntington's disease (HD) is a rare autosomal dominant genetic disease. Patients typically present in middle age and experience motor, cognitive, and psychiatric symptoms. The clinical symptoms of Huntington's disease are complex and changeable, and the patient's condition worsens progressively, and patients usually die 15 to 20 years after the onset of disease. The disease has an insidious onset, slow progression, dance-like movements accompanied by progressive cognition, mental dysfunction and eventually dementia.
The cause is the misexpression of polynucleotide repeats on the huntingtin gene, which affects different molecular pathways and ultimately leads to neurological dysfunction and degeneration. Its pathogenic gene Htt (Huntingtin) encodes Htt protein (Huntingtin protein), and its first exon contains repeated CAG triplet codons. In HD, the number of repeats of this triplet codon is abnormally increased. Individuals with more than 36 CAG repeat triplets are affected. Mutated Htt proteins expressed in the brain lead to neurological deterioration through different molecular mechanisms. The mutated protein not only increases the abnormal function of the protein but also leads to the loss of normal function.
In animal models of HD, treatments have been developed that reduce or prevent the expression of mutant huntingtin (mHTT)1–3. CRISPR/Cas9-mediated gene editing is permanent and can effectively inhibit the expression of mHTT in vivo . Studies have shown that CRISPR/Cas9 has significant potential in alleviating neurological phenotypes in mouse models of neurodegenerative diseases4. However, whether CRISPR/Cas9 editing can be used to edit/correct mutations and ameliorate neurodegenerative effects in patients remains to be verified. Because large mammals are more similar to humans in brain size, development, and brain structure, it is important to evaluate CRISPR/Cas9 safety issues in preclinical studies in large animal models that are biologically and physiologically similar to humans. Indeed, there is growing evidence that CRISPR/Cas9 can be used in non-human primates to model important brain diseases, including Parkinson's disease and autism. Therefore, there is an urgent need to evaluate the safety and therapeutic efficacy of CRISPR/Cas9 in large animals.
Similar to the human brain, the pig brain has extensive cortical gyri, which may confer unique cognitive functions to large mammals. Pigs have been used to model various human diseases. Professor Li Xiaojiang's team at Jinan University's Guangdong-Hong Kong-Macao Central Nervous Regeneration Institute established the HD knock-in (KI) pig 7 in 2018, which can reproduce the selective neurodegeneration seen in the brains of HD patients.
Nature Biomedical Engineering
On February 16, 2023, Professor Li Xiaojiang, Professor Li Shihua, and Research Fellow Yan Sen from the Guangdong-Hong Kong-Macao Central Nervous Regeneration Institute of Jinan University, together with Researcher Lai Liangxue from the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, published a paper titled "Nature Biomedical Engineering" Research paper on "Cas9-mediated replacement" of expanded CAG repeats in a swine model of Huntington's disease. This work uses AAV viral vector to express CRISPR/Cas9 gene editing technology to repair and knock out the mutant gene in the Huntington pig model, evaluates the possible toxicity of CRISPR/Cas9 in the pig brain, and uses HD KI pigs to detect CRISPR/Cas9 Effects on enhancing mHTT and its related phenotypes.
This research work uses AAV9 to target the mutated HTT gene in HD KI pigs to detect the therapeutic effect. The AAV9 virus was stereotactically injected into the cerebral striatum of 6-month-old pigs. The viruses expressing gRNA and Cas9 were mixed at a ratio of 1:2, and 30 μl of the mixed virus (a total of 1-1.5 × 1012 vg) was injected into the pig striatum. Both sides of the body (15 μl per side); gRNA and Cas9 viruses were injected into 3-7 day old piglets through the auricular vein, 2 × 1013 VG/kg, approximately 1.8 × 1013 VG per piglet. Research results show that CRISPR/Cas9-mediated genome editing has no significant toxicity. A single brain stereotaxic injection or intravenous injection of CRSPR/Cas9 and partial replacement of mHTT can effectively reduce neurodegeneration and alleviate neurological phenotypes.
This study proves that CRISPR/Cas9 plays a prominent role in the treatment of large animal models of neurodegenerative diseases and that CRISPR/Cas9 has great potential in treating human neurological diseases.
Large animals, such as dogs, pigs, and non-human primates, are important experimental subjects for disease mechanism research and drug screening. However, achieving gene delivery in large animals requires a large amount of high-quality viral vectors, which are difficult to provide stably with the current conventional 293 production system.
Based on the above needs, Brain Case uses the One-Bac production system based on insect cells SF9 with independent intellectual property rights to launch high-quality and high-volume AAV production services suitable for large animals, stably providing greater than 1E+14 AAV viral vectors to the scientific research community and drug research and development fields.
References:
1. Fyfe, I. Antisense oligonucleotides improve cognitive function in HD model. Nat. Rev. Neurol. 14, 690–691 (2018).
2. Kordasiewicz, H. B. et al. Sustained therapeutic reversal of Huntington’s disease by transient repression of huntingtin synthesis. Neuron 74, 1031–1044 (2012).
3. Southwell, A. L. et al. Huntingtin suppression restores cognitive function in a mouse model of Huntington’s disease. Sci. Transl. Med. 10, eaar3959 (2018).
4. Duan, Y. et al. Brain-wide Cas9-mediated cleavage of a gene causing familial Alzheimer’s disease alleviates amyloid-related pathologies in mice. Nat. Biomed. Eng. 6, 168–180 (2022).
5. Yang, W., Li, S. & Li, X.-J. A CRISPR monkey model unravels a unique function of PINK1 in primate brains. Mol. Neurodegener. 14, 17 (2019).
6. Zhou, Y. et al. Atypical behaviour and connectivity in SHANK3-mutant macaques. Nature 570, 326–331 (2019).
7. Yan, S. et al. A Huntingtin knockin pig model recapitulates features of selective neurodegeneration in Huntington’s disease. Cell 173, 989–1002.e13 (2018).
