Research Concept

In order to realize gene therapy to repair gene mutations that are the root cause of intractable diseases, it is necessary to take various strategies according to type of mutation or disease. In our laboratory, we challenge these issues by applying gene delivery technology using viral and non-viral vectors, and genome editing technology such as CRISPR-Cas9. Here are some of the research projects conducted in the Hotta lab.

CRISPR-Cas9 Genome Editing

CRISPR is a type of DNA cleavage system derived from bacteria, in the right figure gRNA (guide RNA, purple) binds to target DNA (red and orange) to find the target sequence on the genome, Cas9 protein (green) DNA cleavage domain to induce DNA cleavage at the target site. By freely engineer gRNA responsible for DNA binding, DNA damage can be induced at any position on the human genome, to induce deletions or insertion. We aimed to restore the dystrophin protein by using CRISPR-gRNA targeted near the mutation site of the dystrophin gene.

Genome Editing Therapy for Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a severe muscle degeneration disease caused by the loss-of-function mutations in Dystrophin gene on X chromosome. Dystrophin is one of the biggest protein coding genes consist with 2 million bases. Exon skipping to modulate mRNA splicing patterns using antisense oligonucleotide is a promising approach currently tested in clinical trials, however, the effect of antisense oligos is transient. We aim to restore the mutated dystrophin protein using the programmable nucleases, such as CRISPR-Cas9 system, in the patient-derived induced pluripotent stem (iPS) cells as a model.

We first searched the human genome sequence in silico and extracted only a short DNA sequence of 10-16 bases in length on the human genome to create the iGEATs database. By using the iGEATs database, TALEN and CRISPR recognition sequences can be found regions that can specifically target only one place on the human genome. Next, iPS cells were generated from a patient with muscular dystrophy and showed that the mutation of the dystrophin gene can be repaired using three strategies. Furthermore, when we examined the repair efficiency with TALEN and CRISPR, we found that either is effective. Next, we analyzed karyotype of the chromosome, copy number variation of genomic DNA, and base mutation of the protein coding regions in the iPS cells that were repaired, but no significant mutation was found. This result shows that genome editing can be performed with few side effects in iPS cells if TALEN and CRISPR are correctly designed and targeted. Finally, when the original and repaired iPS cells were differentiated into skeletal muscle cells, no expression of dystrophin protein was observed in the original iPS cells, whereas the dystrophin protein was detected in iPS cells after gene repair to the similar level as a healthy person. This result is expected to be a novel method of gene therapy for Duchenne muscular dystrophy.

This study was published in Stem Cell Reports. (Nov 26, 2014)

Li HL, Fujimoto N, Sasakawa N, Shirai S, Ohkame T, Sakuma T, Tanaka M, Amano N, Watanabe A, Sakurai H, Yamamoto T, Yamanaka S, and Hotta A
Precise correction of the Dystrophin gene in Duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9.
Stem Cell Reports, 2015; Vol.4 (1): p143-154. [PubMed link] [Journal link]

Hemophilia gene therapy with DNA transposon vector

Hemophilia A is a congenital bleeding disorder caused by the deficiency of plasma coagulation Factor VIII. Severe hemophiliacs have only a few percentage of Factor VIII activity compared with healthy individuals, and require frequent injections of recombinant Factor VIII to prevent bleeding events. However, this complement therapy is demanding for patient in terms of frequent venous access and costs of recombinant Factor VIII products.

Our research group focuses on development of gene delivery vectors to achieve long-term expression of Factor VIII, since Factor VIII cDNA is too large to be packaged in conventional gene therapy vectors. We envision that establish of liver-like cells from patient's own iPS cells can be genetically engineered to produce high-level of Factor VIII. Such novel approach may have potential to overcome these issues.

We succeeded in delivering a large full-length Factor VIII gene, by utilizing a DNA transposon vector isolated from moth, called as piggyBac vector. In addition, we managed to recover blood coagulation ability in hemophilia A model mice by hydrodynamic injection of the piggyBac vector.

This study was published in PLOS ONE. (Aug 15, 2014)

Matsui H, Fujimoto N, Sasakawa N, Ohinata Y, Shima M, Yamanaka S, Sugimoto M, and Hotta A
Delivery of full-length Factor VIII using a piggyBac transposon vector to correct a mouse model of hemophilia A.
PLOS ONE, 2014; Vol.9 (8): e104957 [PubMed link] [Journal link]

Derivation of human iPS cells with EOS pluripotency reporter

iPS cells can be generated from adult somatic cells by introducing a cocktail of transcriptional factors, and have an enormous potential for future stem cell therapy. However, induction efficiency of iPS cells is still low (~0.02%), and heterogeneous nature of reprogramming makes difficult to control the quality of the iPS cell lines and their differentiation potentials.

When I was in the Ellis lab at Toronto, I have developed a novel selection system for human iPS cells by using a lentiviral vector that specifically express GFP (Green fluorescence protein) gene in pluripotent stem cells. By utilizing Early Transposon (ETn) promoter combined with Oct-4 (= Pou5f1) core enhancer elements, resultant EOS lentiviral vector can express GFP specifically in pluripotent stem cells, but extinguished after differentiation.

When the EOS vector was introduced into human fibroblasts, GFP was not activated as expected. However, after induction of iPS cell reprogramming by a forced expression of the Yamanaka factors (OCT-4, SOX2, KLF4, C-MYC), GFP expression was activated on emerged iPS cell colonies. By selecting for puromycin resistance gene which is under the control of the EOS cassette, only iPS cells can be grown, and enriched the percentage of hESC marker (i.e. TRA-1-81) positive iPSC population up to 70%. We also succeeded to isolate iPS cell lines from Rett Syndrome patient who has neurodevelopmental disorder due to a missence mutation in MeCP2 gene. The EOS vector is not only useful for selecting iPS cells, but also has a potential to utilize for optimizing novel induction methods and for screening small molecules to enhance reprogramming.

This study was published in Nature Methods. (April 26, 2009)

Hotta, A, Cheung, AY, Farra, N, Vijayaragavan, K, Seguin, CA, Draper, JS, Pasceri, P, Maksakova, IA, Mager, DL, Rossant, J, Bhatia, M, Ellis, J.
Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency.
Nature Methods, 2009; Vol.6 (5): p370-376. [PubMed link] [Journal link]