With the increasing number of gene therapy drugs being approved by the federal authorities in the US, Europe and Japan, gene therapy is drawing increasing attention. This recent clinical success has been realized mainly by the development of efficient gene delivery vectors. Adeno-associated virus (AAV) vectors have been used extensively as a stable gene expression vector in vivo and as a vector for delivering donor DNA for gene repair. However, the packaging capacity of AAV vectors is relatively small, and the expression is moderate. In addition to the original CRISPR/Cas9 system, modified versions of Cas9, including base editors and prime editors, have been developed. These editors can precisely modify chromosomal DNA without introducing double-strand breaks and are thus safer than the current CRISPR/Cas9 system with possible widespread applications in the future. However, these new nucleases are too big to be cloned into AAV vectors.
To overcome the limitations associated with AAV vectors, we propose the development of a noble vector that possesses a high cloning capacity and works as an efficient donor vector for gene repair. At present, we have constructed the first version of this vector. The vector efficiently infected a human leukemia cell line and human CD34(+) hematopoietic progenitor cells. In addition, in the human leukemia cell line, the new vector served as an efficient donor vector for gene repair. By the end of fiscal year 2021, we will develop a dual vector system able to accommodate up to 30 kb of therapeutic genes as well as artificial nucleases and also serve as an efficient donor delivery vector for gene repair therapy. In addition, to evaluate the potential of this new system under conditions that closely mimic human applications, a marmoset model of human inherited blood disorder will be used as a target for gene repair therapy using this vector. Together with AAV vectors, this versatile vector is expected to expand the target diseases and future applications for gene therapy, including genome editing.
Figure 1: Development of a dual vector system, which is applicable to strong gene expression and to deliver donor DNA for gene repair.
Figure 2: Research organization
Figure 3: Laboratory scene