Junjiu Huang

Sun Yat-sen University

https://twitter.com/kanzure/status/1068629065941299200

Introduction

Good morning. Thank you for the introduction and for inviting me here. Today I would like to give an overview of basic research about fixing beta-thalassemia in human embryo.

Thalassemia

Thalassemia is a genetic blood disease which was found in 1925. Beta-thalassemia has a high incidence of genetic diseases in the south of China especially in Guangdong, Guangxi and Guizhou province. The carrier rate is between 3-24%. It's the most common genetic disease in my city.

People with thalassemia can get a ... in their body. It can cause damage to their heart, liver and spleen and so on. The patient needs regular blood transfusion until they die. If lucky enough, a few of them can do allogeneic hematopoietic stem cell transplantation.

Genetic mechanism of thalassemia

There are two main types of thalassemia. There's alpha-thalassemia and beta-thalassemia. The skin cell is included in beta-thalassemia.

Gene therapy with lentiglobin by Bluebird Bio

Gene therapy for beta-thalassemia has been studied for more than 30 years. The company Bluebird Bio has completed phase 1 and phase 2 clinicial trials. Clinical approval for this gene therapy may come in 2019 in Europe.

Gene editing

Science: Breakthrough of the year 2012 (TALEN)
Science: Breakthrough of the year 2013 (CRISPR/Cas9)
Nature: Ten people who mattered this year 2013 (Feng Zhang)
Nature: Ten people who mattered this year 2015 (Junjiu Huang)
Science: Breakthrough of the year 2015 (CRISPR/Cas9)
Nature: The science events that shaped 2015 (CRISPR/Cas9)
Nature: Ten people who mattered this year 2017 (David Liu)
Science: Breakthrough of the year 2017 (pinpoint gene editing)
Nature: The science events that shaped 2017 (Human embryo editing)

Gene editing technique .. to modify DNA sequence... genetic mutations. This is important for gene therapy.

Treat beta-thalassemia with gene editing in HSCs

We can isolate CD34+ stem cells and modify the mutation site. We make a suspension of the stem cells and then do transplantation.

Fixing beta-thalassemia in somatic cells

CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells (2016)
Direct promoter repression by BCL11A controls the fetal to adult hemoglobin switch (2018)

.. by precisely repairing the mutation site, or repression of BCL11A to control hemoglobin.

How to get a healthy baby for patients?

Another important issue for the genetic disease patient is how to get a healthy baby for them, especially when both parents are carriers. Only 25% of their embryos are normal. 50% are carriers.

Third generation test-tube baby technology

Tangential to this, .... in our first facility hospital which was formed in 1989. In our IVF center, we use third-generation test tube baby technology called PGT (preimplantation genetic testing) to help beta-thalassemia/DMD patients since the year 2000.

In some cases, it's hard to get normal embryos. We we ask, can we repair or block inherited mutations in human embryos?

Mouse model research

Using gene targeting and gene editing techniques, I have produced many different types of genetically modified mice for basic research in the past more than 10 years.

Correction of a gene mutation in mouse zyogte

Correction of a genetic disease in mouse via use of CRISPR-Cas9 (2013)

In 2013, Jinsong Li used CRISPR-Cas9 to fix the genetic mutation causing thalassemia in mouse embryos and get healthy pumps. What risks are there in fixing beta-thalassemia in human embryos using CRISPR-Cas9?

CRISPR/Cas9-mediated editing of HBB gene in human cells

So we start our study in about 2014. At the beginning, we used the cell model to screen the best and also the single strand DNA to repair the mutation size in cell model.

CRISPR/Cas9-mediated editing of HBB gene in human tripronuclear zygotes

After we get the best ssDNA, we inject those into tripronuclear zygotes. They are the best models for studying. Surprisingly, we found a high rate of endogenous repair, recombination based on HBD. It's about 25%.

We also found off-target effect in human tripronuclear embryos.

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes (2015)

There are still many risks for gene editing in human embryos with CRISPR-Cas9 such as NHEJ (nonhomologous end joining), mosaicism, off-target mutations, and using endogenous genes as templates.

Correction of a pathogenic gene mutation in human embryos (2017)

In 2017, Mitalipov's team discovered that using normal human embryos, he could use ... as template to repair the mutant allele after gene editing. NHEJ, mosaicism and off-target problems are not yet solved.

In 2015, after the first tripronuclear with CRISPR-Cas9, it was not the suitable tool to use at that point.

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage (2016)

So in 2016, David Liu has published a paper about base editor which does C -> T and G -> A.

Effective gene editing by high-fidelity base editor 2 in mouse zygotes (2017)
Editing base in mouse model (Hanyi Wang)

We tested the base editor in mouse embryo. We found that we can generate homozygous founder mice with the base editor, which means 100% editing in mouse embryo.

In mice, beta-thalassemia is caused by poly mutation. There's more than 100 mutants in various genes. Can we repair beta-thalassemia in human embryos with the base editor?

We checked beta-thalassemia in the hospital, at Memorial Hospital, Sun Yat-sen University. This analysis was made by Jianpei Fang (2011-2016 analysis). We found that there's a mutation in -28M/N(A-to-G) which is one of the top 3 mutations in beta-thalassemia and a potential target site for the base editor system.

Correcting HBB -28 (A to G) mutation in human cell line

APOBEC-XTEN-dCas9(A840H)-UGI

So we first constructed a human cell line with this mutation. So we screened, to see which one can mediate the precise repair of the HBB -28 error in cells. After that, we also isolated skin fibroblast cell from a homozygous -28 patient. We tried that system in these cells too. As you can see here, about 20% homozygous cells are repaired to heterozygous. There is still some unwanted repair.

Precise editing a single base with YEE-BE3

Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions (2017)

In 2017, David Liu published another paper of another version of the base editor 3 (BE3) called YEE-BE3 which can site-edit a single base.

We used this base editor and constructed a homozygous human embryo, and after we inject YEE-BE3 into the human embryo we found about 20% blastomere cells have been repaired to heterozygote. In this experiment, we did not find any off-target effects.

The world's first report of human embryos altered by base editor

Correction of beta-thalassemia mutant by base editor in human embryos (2017) (Junjiu Huang)

"This first study was a successful proof of principle that the base-editing technique can be used to correct a disease mutation in a human embryo." - Nature 2017

Adenosine base editor (ABE)

Programmable base editing of A-T to G-C in genomic DNA without DNA cleavage

So now David Liu has published another version, an ABE system for changing A-T to G-C. As he showed us yesterday, using a combined system, maybe we can repair more than 50% of the mutation sites.

Also yesterday, Professor Yongfei has published a paper on biorxiv (the bio list) so that the target of the base editor... so there's more question to do.

Here I want to say what I have done is under the guidelines of China and is also consistent with the guidelines from the last conference.

Condemnation statement

Here I also want to post an official statement which is from our Committee of Genomic Editing, Genetics Society of China, Chinese Society for Stem Cell Research. We strongly condemn any application of gene editing on human embryos for reproductive purposes. Such intervention is against the law, regulation, and medical ethics of China. Moreover, it violates internationally accepted ethical principles regulating human experimentation and human rights laws.

Thank you. That's all. Thank you.