Grants:

2011 GRF (PI); 2012 ECS (PI); 2012 NSFC (PI); 2013 GRF (PI); 2014 GRF (PI); 2015 Scheme-B (PI); 2016 BTF (PI); 2017 BTF (PI); 2018 GRF (PI); 2019 GRF (PI); 2020 GRF (PI)

Large group grants: 2012 TBR (co-I); 2014 973-grant (co-I); 2015 CRF (co-I); 2017 TBR (co-I); 2018 TBR (Co-I); 2021 CRF (Co-PI); 2021 ITF (Co-PI)

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Student Awards:

2014 Reaching Out Award to HU Jiabiao
2016 Reaching Out Award to HE Xiangjun
2016 Postgraduate Research Output Award to HE Xiangjun

2017 SBS Postgraduate Research Day 2017 The Third Prize to ZHANG Chenzi                       

2018 Third prize in the oral presentation of 6th Cross-strait Symposium on Biomedical Sciences at Kunming Institute of Zoology to ZHANG Chenzi                                    

2019 2017-2018 Staff Scholarship for BSc in Biomedical Sciences Programme to WANG Jingyi

2019 SBS Postgraduate Research Day 2018 The Third Prize to ZHANG Chenzi

2019 SBS Postgraduate Research Day 2018 The Top 10 Poster Award to ZHANG Chenzi   

2021 Best Presentation - Sliver Award in Poster Presentation 2021 to WONG Hoi Ting   

2021 Best Presentation - Sliver Award in Poster Presentation 2021 to LEUNG Sum Yin    

2021 SBS Postgraduate Research Day 2021 Second Prize in the Oral Presentation to ZHANG Zhenjie     

2021 SBS Postgraduate Research Day 2021 Most Popular Poster Prize to ZHANG Zhenjie      

2021 SBS Postgraduate Research Day 2021 Most Popular Poster Prize to WANG Jingyi    

2022 2020-2021 Staff Scholarship for BSc in Biomedical Sciences Programme to TSANG Kin Ching    

2022 Best Presentation Award in Capstone Research Project Presentation Day 2022 to TSANG Kin Ching    

2022 Best Presentation – Bronze Award in Poster Presentation to CHEUNG Lok Lam   

2022 Prof. Leung Po Sing Scholarship 2021/22 to WANG Jingyi   

2022 “2022粵港再生醫學研究生學術交流”大會口頭報告二等獎 to ZHANG Siqi    

2022 “2022粵港再生醫學研究生學術交流”大會墻報展示三等獎 to WEI Junkang  

2022 CRMH Research Day 2022 – Best Oral Presentation Award to ZHANG Zhenjie 

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Highlights of Research:

1.       Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair

CRISPR/Cas9-introduced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) repair mechanisms. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells, including pluripotent human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) which possess big potentials in regenerative medicine. However, most of studies focused on HDR-based strategies which were proven inefficient. We constructed a universal reporter system and systematically investigate into the potentials of both HDR and NHEJ repair in mediating CRISPR/Cas9-induced reporter integration. Here, we found that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells.  

He et al., 2016, Nucleic Acids Research

2.       Developing safer AAV-CRISPR gene knock-in therapy for the treatment of hemophilia B in human

Recombinant adenovirus associated virus (AAV) vector-based delivery of CRISPR/Cas9 (AAV-CRISPR) has shown promising potentials in preclinical models to efficiently insert therapeutic gene sequences in somatic tissues. However, the doses of AAV input required for effective targeting were prohibitively high which posed serious risk of toxicity. We performed AAV-CRISPR mediated homology independent knock-in at the proximal mAlb 3'UTR and demonstrated that single dose of AAVs enabled long-term integration and expression of hF9 transgene in both adult and neonatal hemophilia B mice (mF9 -/-), as evidenced by high levels of circulating hFIX and restored hemostasis during the entire 48-week observation period. The germline genomes from edited mice were free of modification. No evident changes at transcriptome level were associated with AAV-CRISPR-mediated hF9 knock-in, and no off-target editing events were detected at the top 10 in silico-predicted sites. Furthermore, we demonstrated that hemostasis correction can be efficiently achieved with a much lower AAV dose (2 × 10⁹ vg/neonate and 1.6 × 10¹⁰ vg/adult mouse) through liver-specific gene knock-in using hyperactive hF9^R338L variant. The serum antibodies against Cas9 and AAV in the neonatal mice receiving low-dose AAV-CRISPR were negligible, which lent support to the development of AAV-CRISPR mediated somatic knock-in for treating inherited diseases.

He X. Zhang Z. et al., 2022, Nature Communications

3.       Generation of universal CAR-T cells through one-step targeting

T-cells engineered to express chimeric antigen receptor (CARs) represents an innovative and revolutionizing approach for cancer immunotherapy. In the clinic, CAR-T therapies targeting CD19 and BCMA have achieved overall response rates (ORR) of 73% and 82%, respectively. However, the remaining 20-30% of patients still experienced relapsed or refractory (R/R) diseases, which averted the eradication of malignant cells after CAR-T therapy.

The current FDA-approved CAR-T products are all based on autologous transplantation, for which the manufacturing processes are exorbitantly expensive, and varied quantity and quality of starting T-cells obtained from patients significantly influence the treatment outcomes. To overcome the inherent limitation of autologous CAR-T products, we have initiated a research project to develop universal allogeneic CAR-T cells (UCAR-T) engineered through AAV-delivered CRISPR/Cas9 gene editing to reduce the risk of triggering GvHD (supported by ITF Ref. ITS/153/20FP). Our research has yielded promising results in manufacturing large numbers of effective U-CAR-T cells and demonstrated excellent efficacy against cancer and tolerability using leukaemia xenograft mouse models.

The development of “U-CAR-T” is anticipated to bypass the dependency on autologous patient-specific T cells to broaden the range of patients to receive treatment, particularly benefit pediatric and heavily treated patients.

Zhang C. Ngan CC. et al. unpublished

4.       Integrative view of pluripotency through bioinformatics tools

Nowadays, bioinformatics plays a crucial role in ESCs/iPSCs studies by providing comprehensive interpretation through integrating the massive information from multiple research platforms, such as microarray and next-generation sequencing (NGS) data. In our lab, we use bioinformatics tools to integrate the datasets of microarray, ChIP-seq, RNA-seq, as well as methyl-seq. Intensive knowledge can be acquired for understanding the underlying transcription regulations, gene expressions, as well as epigenetic modifications during stem cell differentiation and reprogramming processes. The research output can provide valuable information to understand the molecular mechanisms of pluripotent network in embryonic stem cells (Wang et al. unpublished).

Wang et al. unpublished

Projects in near future:

 AAV-CRISPR strategies for gene and cell therapy

 Study of hepatic differentiation from human ESC/iPSCs

 Molecular mechanism underlying the lineage specification and cell fate control 

 Study on liver stem cells and their applications in the treatment of end-stage liver disease