Weiyu Zhao

Engineering CRISPR–Cpf1 crRNAs and mRNAs to maximize genome editing efficiency

Cpf1, a type-V CRISPR-Cas effector endonuclease, exhibits gene-editing activity in human cells through a single RNA-guided approach. Here, we report the design and assessment of an array of 42 types of engineered Acidaminococcus sp. Cpf1 (AsCpf1) CRISPR RNAs (crRNAs) and 5 types of AsCpf1 mRNAs, and show that the top-performing modified crRNA (cr3′5F, containing five 2′-fluoro ribose at the 3′ termini) and AsCpf1 mRNA (full ψ-modification) improved gene-cutting efficiency by, respectively, 127% and 177%, with respect to unmodified crRNA and plasmid-encoding AsCpf1. We also show that the combination of cr3′5F and ψ-modified AsCpf1 or Lachnospiraceae bacterium Cpf1 (LbCpf1) mRNAs augmented gene-cutting efficiency by over 300% with respect to the same control, and discovered that 11 out of 16 crRNAs from Cpf1 orthologs enabled genome editing in the presence of AsCpf1. Engineered CRISPR-Cpf1 systems should facilitate a broad range of genome editing applications.

Biodegradable Amino-Ester Nanomaterials for Cas9 mRNA Delivery in Vitro and in Vivo

Efficient and safe delivery of the CRISPR/Cas system is one of the key challenges for genome-editing applications in humans. Herein, we designed and synthesized a series of biodegradable lipidlike compounds containing ester groups for the delivery of mRNA-encoding Cas9. Two lead materials, termed N-methyl-1,3-propanediamine (MPA)-A and MPA-Ab, showed a tunable rate of biodegradation. MPA-A with linear ester chains was degraded dramatically faster than MPA-Ab with branched ester chains in the presence of esterase or in wild-type mice. Most importantly, MPA-A and MPA-Ab demonstrated efficient delivery of Cas9 mRNA both in vitro and in vivo. Consequently, these biodegradable lipidlike nanomaterials merit further development as genome-editing delivery tools for biological and therapeutic applications.

Design, synthesis and evaluation of anti-CD123 antibody drug conjugates

Leukemia stem cells (LSCs) account for the development of drug resistance and increased recurrence rate in acute myeloid leukemia (AML) patients. Targeted drug delivery to leukemia stem cells remains a major challenge in AML chemotherapy. Overexpressed interleukin-3 receptor alpha chain, CD123, on the surface of leukemia stem cells was reported to be a potential target in AML treatment. Here, we designed and developed an antibody drug conjugate (CD123-CPT) by integrating anti-CD123 antibody with a chemotherapeutic agent, Camptothecin (CPT), via a disulfide linker. The linker is biodegradable in the presence of Glutathione (GSH, an endogenous component in cells), which leads to release of CPT. Anti-CD123 antibody conjugates showed significant higher cellular uptake in CD123-overexpressed tumor cells. More importantly, CD123-CPT demonstrated potent inhibitory effects on CD123-overexpressed tumor cells. Consequently, these results provide a promising targeted chemotherapeutical strategy for AML treatment.

Co-delivery of mRNA and SPIONs through amino-ester nanomaterials

Nanoparticles have been widely explored for combined therapeutic and diagnostic applications. For example, lipid-based nanoparticles have been used to encapsulate multiple types of agents and achieve multi-functions. Herein, we enabled a co-delivery of mRNA molecules and superparamagnetic iron oxide nanoparticles (SPIONs) by using an amino-ester lipid-like nanomaterial. An orthogonal experimental design was used to identify the optimal formulation. The optimal formulation, MPA-Ab-8 LLNs, not only showed high encapsulation of both mRNA and SPIONs, but also increased the r2 relaxivity of SPIONs by more than 1.5-fold in vitro. MPA-Ab-8 LLNs effectively delivered mRNA and SPIONs into cells, and consequently induced high protein expression as well as strong MRI contrast. Consistent herewith, we observed both mRNA-mediated protein expression and an evident negative contrast enhancement of MRI signal in mice. In conclusion, amino-ester nanomaterials demonstrate great potential as delivery vehicles for theranostic applications.

Phosphorothioate-modified DNA oligonucleotides inactivate CRISPR-Cpf1 mediated genome editing

CRISPR-Cpf1, a microbial adaptive immune system discovered from Prevotella and Francisella 1, employs a single-stranded CRISPR RNA (crRNA) to induce double stranded DNA breaks1. To modulate genome editing activity of Cpf1 in human cells, we designed a series of crRNA variants including DNA-crRNA and RNA-crRNA duplexes, and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1 mediated gene editing. More importantly, without prehybridization, this PS-modified DNA was able to regulate Cpf1 activity in a time-and dose-dependent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder the binding between Cpf1-crRNA complex and target DNA substrate. Consequently, phosphorothioate-modified DNA oligonucleotides provide a tunable platform to inactivate Cpf1 mediated genome editing.

GlcNAc Conjugated Atorvastatin with Enhanced Water Solubility and Cellular Internalization

Targeting ligands facilitate cell specific drug delivery and improve pharmaceutical properties. Herein, we designed two ligand drug conjugates by conjugating GlcNAc (N-acetylglucosamine) with atorvastatin. These two conjugates, termed G-AT and G-K-AT, exhibited enhanced water solubility and cellular uptake. Moreover, both G-AT and G-K-AT were able to release atorvastatin and consequently achieve significant inhibition against 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase.