Gene therapy, or the manipulation of genes to correct disease, has been a buzzword since it was first tested in humans in 1990. Viral and non-viral vectors such as liposomes have been used to deliver DNA into target cells. Alternatively, naked DNA vectors such as plasmids and minicircles have been explored as gene delivery vehicles. However, DNA in these vectors is usually silenced in primary cells (cells taken from the body and cultured, vs cell lines).
That’s where the work of Assistant Professor Volker Patzel of the Department of Microbiology comes into play. Asst Prof Patzel is no stranger to nucleic acids, having researched RNA since he was a PhD student at the German Cancer Research Center in Heidelberg. Following a postdoc at the Max-Planck Institutes, he joined NUS in 2009.
Asst Prof Patzel’s research at NUS involves naked dumbbell-shaped vectors. Consisting of a closed DNA loop resembling a dumbbell, these vectors lend themselves to gene delivery for several reasons (see Facts Box). The Patzel lab is using the vectors to manipulate gene expression in three ways: 1) RNA-guided genome editing, 2) suicide gene therapy, and 3) somatic cell reprogramming (see Figure).
The first project involves editing the genome using the innovative CRISPR-Cas9 system. The Patzel lab has adapted the dumbbell-shaped vector to carry the genes for CRISPR-Cas9. Once inside the nucleus, a small guide RNA directs the Cas9 enzyme to a target DNA sequence, where Cas9 cuts both DNA strands. The double-stranded break triggers mechanisms in the cell that repair the cut. Thanks to this powerful technology, a faulty gene that causes a disease can be repaired with surgical precision. Mutations can also be introduced during editing to study their effects on gen e function or to destroy aberrant genes. The team is optimizing the guide RNA structure for greater efficiency and using new guide RNAs to target different disease genes.
The Patzel lab is also exploring the use of dumbbell-shaped vectors that carry “death signals” for suicide gene therapy. When these molecular grenades are introduced into cancer cells or virus-infected cells, they fuse the death signal to target cell messages through a process called RNA trans-splicing, causing the cells to self-destruct.
The third research focus uses the dumbbell-shaped vectors to deliver microRNAs (miRNAs) into cells to inhibit specific genes and reprogram the cells. For example, somatic cells (any cell in the body besides the sperm and egg) can be reprogrammed to produce induced pluripotent stem cells for research and transplantation uses.
Asst Prof Patzel continues to improve the dumbbell technology. He is exploring putting cell-penetrating peptides on the vectors to further ease their entry into cells. He also hopes to collaborate with clinicians to study the application of dumbbell-shaped vectors in humans.
FACTS
Advantages of Dumbbell-Shaped Vectors for Gene Delivery
- Closed loop structure increases resistance to enzymatic breakdown
- Small size (contains minimal DNA sequence) facilitates cell entry
- Once inside cell, flexible ends aid passage through nuclear pore complex into nucleus
- Lack extra-genic sequences, thus do not trigger transgene silencing in primary cells
- Option to link helper functions such as fluorescent labels to the single-stranded loops
Figure. Dumbbell-Shaped Vector Applications in the Patzel Lab
References
- Yu H, Jiang X, Tan KT, Hang L, Patzel V. Efficient production of superior dumbbell-shaped DNA minimal vec tors for small hairpin RNA expression. Nucleic Acids Res. 2015 Jun 11. [Epub ahead of print].
- Kobelt, D, Aumann J, Schmidt M, Wittig B, Fichtner I, Behrens D, et al. Preclinical study on combined chemo- and nonviral gene therapy for sensitization of melanoma using a human TNF-alpha expressing MIDGE DNA vector. Mol Oncol. 2014;8:609-619.
- Jennifer Doudna (UC Berkeley / HHMI): Genome Engineering with CRISPR-Cas9. Available at: https://www.youtube.com/watch?v=SuAxDVBt7kQ.