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Viral hepatitis B is a common infectious disease affecting the world, the cause of which is hepatitis B virus (HBV) infection and induced autoimmune reaction. When infected, HBV's rcDNA is converted into cccDNA, and its genes may also be integrated into the human genome. HBV cannot be eradicated with traditional chemical treatments, but it is possible with the safety-enhancing CRISPR-Cas gene editing tool.
This project aims to treat hepatitis B with CRISPR-Cas gene editing technology, take HBV HBsAg gene as the targeted sequence, construct Cas12a nuclease MAD7 mutation library, combine high-throughput screening methods to obtain high-precision and high-activity nuclease variants, and use customized LNP delivery system. Development of hepato-targeted hepatitis B gene therapy.
Viral hepatitis type B (HBV) is a common infectious disease caused by Hepatitis B virus (HBV) infection, mainly liver lesions. Who estimates that 296 million people are living with chronic hepatitis B infection and 1.5 million people are newly infected with hepatitis B each year (2019). In China, the number of new hepatitis B patients each year accounts for one-third of all infectious diseases.
The pathogenesis of hepatitis B is that after the relaxed DNA (rcDNA) of HBV invades the nucleus, it is repaired into covalent closed circular DNA (cccDNA), and is integrated into the human genome with the Hepatitis B surface antigen (HBsAg) gene. HBsAg is expressed and attached to the surface of liver cells, allowing them to be attacked by the patient's own immune system. Long-term damage to the immune response of liver cells may lead to fatal diseases such as cirrhosis, liver cancer (HCC), or systemic complications such as hepatogenic diabetes. Therefore, early effective treatment of hepatitis B patients is very critical.
At present, hepatitis B is treated with drugs of interferons (mainly pegylated interferon alpha) and nucleoside (acid) analogues (lamivudine, Tibivudine, etc.). They inhibit HBV DNA replication, thereby reducing clinical symptoms in patients. However, these two types of drugs can not completely eliminate HBV, long-term drug use will increase the cost of treatment, and the virus will become resistant. Therefore, it is necessary to develop a one-time cure for hepatitis B.
Based on the physiological characteristics of HBV infected cells, HBV genes can be knocked out by gene editing technology, which can realize the complete elimination of HBV.
The CRISPR-Cas9 system can effectively cut the cccDNA of HBV, thereby inhibiting viral replication [1]. However, previous studies have mostly relied on sustained expression of CRISPR-Cas9 to inhibit HBV, and persistent overexpression of the gene-editing complex increases off-target rates and toxicity. Recent studies have shown [2-6] that the existing CRISPR-Cas9 technology can eradicate more than 98% of HBV cccDNA by means of short delivery of RNPs, combined with isiRNA and interferon, but it also needs to remove the HBsAg gene that has been integrated into the human genome to protect liver cells from immune system attack. This requires our gene-editing tools to be particularly precise and safe.
There is a certain probability that CRISPR-Cas system will misbind with non-target DNA sequences during gene editing, causing unexpected gene mutations, that is, off-target effects, which affect the accuracy and safety of gene editing [7]. Therefore, how to improve targeting specificity and reduce miss rate by modifying Cas protein structure has always been the focus of current research [8-10]. Through rational or irrational means, researchers have successfully developed many mutant Cas nucleases with low off-target rates, and explored many amino acid sites that affect the specificity of Cas nucleases [11-13]. We note that Antonio Casini's team used the Saccharomyces cerevisiae reporter strain to design a two-plasmid screening system for efficient and precise screening of mutant Cas9 nucleases derived from random mutations. By combining mutations, the researchers succeeded in obtaining evoCas9, which is 9 times more accurate than wild-type Cas9 [16]. This evolution-like modification is called directed evolution and has become one of the mainstream ideas for modifying Cas proteins. However, Cas proteins modified by directed evolution are still unable to meet the gene-editing accuracy required for clinical trials.
Cas9 requires tracrRNA and crRNA for cleavage, resulting in blunt ends. Our modified object, MAD7 nuclease, is a Cas12a. Compared with Cas9, Cas12a does not require tracrRNA and HNH domains, and the crRNA of Cas12a is significantly shorter than Cas9, so Cas12a has a smaller molecular weight. Cas12a cleavage produces sticky ends and has the ability to perform non-specific cleavage on single-stranded DNA, effectively clearing HBV rcDNA. Compared with the "NGG" PAM sequence of spCas9, the PAM sequence of MAD7 is "YTTN", which has a wider target range. At present, the transformation of MAD7 is less, and there is still a great potential for structural transformation and performance improvement.
In order to improve the safety of hepatitis B gene editing therapy, we propose the following protocols:
Construct rational MAD7 mutation library by random mutation. At the same time, a rational MAD7 mutation library was constructed using site-specific mutation design according to the dry laboratory prediction results. HBsAg gene was combined with sacB gene and other resistance genes to construct a high-throughput and high-precision screening system for multiple plasmids. The screening system was transformed into Escherichia coli and the MAD7 mutation library was screened in Escherichia coli. The selected high-precision and high-activity nuclease mutants were sequenced to determine the mutation location. On this basis, the highly specific MAD7 variant was constructed by combining combination mutation and saturation mutation. According to the high-precision and high-activity MAD7 gene synthesized mRNA obtained by us, the LNP delivery system is used to target the liver for efficient delivery, so that high-precision hepatitis B gene editing therapy can be achieved.
We hope that this project will bring hope for a cure for hepatitis B and lay the foundation for gene editing to treat the disease.
[1] Revill P A, Chisari F V, Block J M, Dandri M, Gehring A J, Guo H, Hu J, Kramvis A, Lampertico P, Janssen, H L A, et al. A global scientific strategy to cure hepatitis B [J]. The Lancet Gastroenterology & Hepatology, 2019, 4(19):545-558.
[2] Kostyushev D, Kostyusheva A, Brezgin S, et al. Depleting hepatitis B virus relaxed circular DNA is necessary for resolution of infection by CRISPR/Cas9 [J]. Molecular Therapy - Nucleic Acids, 2023, 31(17):482-493.
[3] Cao Z, Liu Y, Ma L, et al. A potent hepatitis B surface antigen response in subjects with inactive hepatitis B surface antigen carrier treated with pegylated-interferon alpha [J]. Hepatology, 2017, 66(4):1058-1066.
[4] Shan Q, Baltes N J, Atkins P, et al. ZFN, TALEN and CRISPR-Cas9 mediated homology directed gene insertion in Arabidopsis: A disconnect between somatic and germinal cells [J]. Journal of Genetics and Genomics, 2018, 45(12):681-684.
[5] Hannah S J Choi, Margo J H, van Campenhout, Anneke J van Vuuren, et al. Ultra-Long-term Follow-up of Interferon Alfa Treatment for HBeAg-Positive Chronic Hepatitis B Virus Infection [J]. Clinical Gastroenterology and Hepatology, 2021, 19(9):1933-1940.e1.
[6] Lee S K, Kwon J H, Lee S W, et al. Sustained off therapy response after peglyated interferon favours functional cure and no disease progression in chronic hepatitis B [J]. Liver International, 2021, 41(2):288-294.
[7] Peng R, Lin G, Li J. Potential pitfalls of CRISPR/Cas9-mediated genome editing [J]. FEBS Letters, 2016, 283(7):1218-1231.
[8] Vakulskas C A, Dever D P, Rettig G R, et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells [J]. Nature Medicine, 2018, 24(8):1216-1224.
[9] Lee J K, Jeong E, Lee J, et al. Directed evolution of CRISPR-Cas9 to increase its specificity [J]. Nature Communications, 2018, 9(1):30-48.
[10] Gao L, Cox D B T, Yan W X, et al. Engineered Cpf1 variants with altered PAM specificities [J]. Nature Biotechnology, 2017, 35(8):789-792.
[11] Kleinstiver B P, Pattanayak V, Prew M S, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome wide off-target effects [J]. Nature, 2016, 529(7587):490-495.
[12] Slaymaker I M, Gao L, Zetsche B, et al. Rationally engineered Cas9 nucleases with improved specificity [J]. Science, 2016, 351(6268):84-88.
[13] Casini A, Olivieri M, Petris G, et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast [J]. Nature Biotechnology, 2018, 36(3):265-271.
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