State-of-the-art human gene therapy: part I. Gene delivery technologies

Abstract

Safe and effective gene delivery is a prerequisite for successful gene therapy. In the early age of human gene therapy, setbacks due to problematic gene delivery vehicles plagued the exciting therapeutic outcome. However, gene delivery technologies rapidly evolved ever since. With the advancement of gene delivery techniques, gene therapy clinical trials surged during the past decade. As the first gene therapy product (Glybera) has obtained regulatory approval and reached clinic, human gene therapy finally realized the promise that genes can be medicines. The diverse gene delivery techniques available today have laid the foundation for gene therapy applications in treating a wide range of human diseases. Some of the most urgent unmet medical needs, such as cancer and pandemic infectious diseases, have been tackled by gene therapy strategies with promising results. Furthermore, combining gene transfer with other breakthroughs in biomedical research and novel biotechnologies opened new avenues for gene therapy. Such innovative therapeutic strategies are unthinkable until now, and are expected to be revolutionary. In part I of this review, we introduced recent development of non-viral and viral gene delivery technology platforms. As cell-based gene therapy blossomed, we also summarized the diverse types of cells and vectors employed in ex vivo gene transfer. Finally, challenges in current gene delivery technologies for human use were discussed.

Figure 1

Gene delivery vectors. A therapeutic transgene expression cassette can be carried by a DNA vector (left) or a viral vector (right) for delivery.

Figure 2

Viral vector engineering. A wild type viral genome contains viral genes (red, orange and gray) and sequences that serve as packaging signal (blue). Removing most of the viral genes creates space for a transgene expression cassette (green) and minimizes virulence.

Figure 3

An example of viral vector production process. Production of recombinant adeno-associated virus (rAAV) is shown. Four components are essential: (1) a cis plasmid carrying a recombinant viral genome that contains a therapeutic gene cassette (green) and packaging signals (blue), (2) a trans plasmid carrying genes encoding viral capsid proteins (Cap) and replication proteins (Rep), (3) a helper plasmid carrying some genes from adenovirus, and (4) a stable producer cell line expressing the adenoviral protein E1. Co-transfection of the three plasmids into the producer cell line leads to the expression of the carried genes (orange and purple). The adenoviral genes carried on the producer cell chromosome and pHelper facilitate Rep expression. Rep recognizes the packaging signal sequences (blue), and excises the rAAV genome from pCis. With the help of some adenoviral proteins, the released rAAV genome and Cap are assembled into rAAV particles, which are further purified. Black solid arrows: co-transfection. Black dashed arrows: gene expression. Gray solid arrows: assembly line of rAAV particles. Gray dashed arrows: roles of the adenoviral proteins (Gao & Sena-Esteves, 2012). For production of other viral vectors, refer to Vannucci et al., 2013.

Figure 4

CAR-armed T cell in cancer gene therapy. Isolated T cell population is genetically modified by ex vivo gene transfer to express CAR on cell surface. After the CAR-armed T cell is reinfused into human body, the CAR recognizes specific molecule on the tumor cell surface. The interaction triggers a cascade of cell signaling event (red bolt), and eventually allows the T cell to kill the tumor cell.