Artificial Chromosome Vector

tech_vecter

Artificial Chromosome Vector

 

Artificial chromosome vectors were developed by Professor Mitsuo Oshimura et al. in Tottori University Graduate School of Medicine by deleting gene regions unnecessary for the maintenance and division of chromosomes in the cell from human or mouse chromosomes. They are called HAC (human artificial chromosomes: artificial chromosomes derived from human chromosomes) vector or MAC (mouse artificial chromosomes: artificial chromosomes derived from mouse chromosomes) vector.

Since these artificial chromosome vectors inherit the properties of chromosomes, they have quite different properties from conventional plasmid/viral vectors.

In the method of constructing a stable gene expression cell line from a plasmid/viral vector or the like, it was necessary to insert the vector into which the desired foreign gene was integrated on the host chromosome in order to maintain it. During this, the vector was randomly inserted on the host chromosome, and the copy number was also variable, which made it difficult to control.

<Problems with the conventional method (plasmid/viral vector)>

  1. Restriction on DNA size to be transfected
  2. Destruction of host genes by insertion of foreign genes
  3. Changes in properties of cells, such as malignant transformation by insertion of foreign genes
  4. Difference in expression level between clones due to difference in insertion position and copy number
  5. Gene silencing by subculture

Since the artificial chromosome vector is retained independently of the host chromosome in the host cell line, the above problems can be solved.

Construction method of HAC (human artificial chromosome) vector

vector_1

References:Y. Kazuki et al., Gene Therapy (2011) 18 384-393

Features of artificial chromosome vector

 

Artificial chromosome vectors have the following advantages compared to conventional vectors. Use for various applications is expected, such as the generation of a stable gene expression cell line, preparation of a transgenic animal, and construction of high protein producing cells.
vector_2
vector_3

Experimental animal production with advanced techniques

 

We produced chromosome-transferred mice utilizing the chromosome engineering technology we have cultivated up to now. In addition, genetic modifications using mouse ES cells, chimeric mouse production, etc., are available.

Trans-chromosomic mouse

We produced trans-chromosomic mice with artificial chromosome vectors. The artificial chromosome vectors have the characteristics, such as being capable of being transmitted to offspring through the germline and not restricting the size of a gene that can be loaded.

By generating a trans-chromosomic mouse with artificial chromosome vectors, it is possible to create a genetically modified mouse containing a huge foreign gene that could not be generated by the conventional knock-in method, and express genes in a tissue-specific and time-specific manner by loading specific control areas. It is also possible to control the expression level by a copy number.

As an example of trans-chromosomic mice, we have succeeded in developing mice transfected with the human CYP3A gene cluster known to be involved in drug metabolism.

Method of creating trans-chromosomic mouse

vector_4

Comparison between in vitro  and in vivo

Artificial chromosome vectors can be transferred not only to various cells by the microcell-mediated chromosome transfer but also they can be maintained stably.

Cells for in vitro evaluation and mouse specimens for in vivo evaluation can be created with artificial chromosome vectors and compared with each other. It is also possible to compare in vitro and in vivo by transplanting disease model cells to an immunodeficient mouse.

Comparison between in vitro and in vivo evaluation system with artificial chromosome vectors

vector_5

Transgenic mouse/knock-in mouse

We also provided development services for ordinary transgenic mice/knock-in mice. Chromosomal analysis, our specialty, makes it possible to prepare high-quality transgenic mice by analyzing the karyotypes of ES cells, identifying insertion positions of foreign genes, and checking the copy number.

Model animal for human drug metabolism

 

In the drug development process, testing pharmacokinetics and safety at an early stage is considered significant to improve development efficiency. However, because there are species differences in the drug-metabolizing enzymes between humans and laboratory animals, such as mice and rats, dropping out often occurs at the stage of clinical trials.

Among drug-metabolizing enzymes, CYP3A is known to be related to the metabolism of about 40% of the drugs on the market, and humanization was desired. However, the human CYP3A gene group (CYP3A5, CYP3A7, CYP3A4, and CYP3A43) exists as a cluster of approximately 700 kb on chromosome 7, making it difficult to humanize the whole cluster simultaneously with existing techniques.
In addition, when gene transfer is performed, a forced expression system by foreign promoters is common, but there was also a problem that it was difficult to approximate tissue-specific or time-specific expression closer to humans.

By applying artificial chromosome vector technology, which is our basic technology, we succeeded in loading not only four types of the CYP3A gene but also an endogenous CYP3A promoter region.

As a result, tissue-specific and time-specific gene expression became possible, and various properties not found in the other model mice for drug metabolism were successfully acquired.

Construction of humanized CYP3A mouse

tech_02_01_image

The region containing the human CYP3A gene group was translocated and cloned on a HAC vector by site-specific recombination after a fragment derived from human chromosome 7 with the loxP sequence inserted and the HAC vector were allowed to coexist in the same cell. FISH analysis showed that the human CYP3A gene group (green) was translocated on the artificial chromosome vector (red).
vector_7

The constructed CYP3A-HAC vector was transferred to mouse ES cells by the microcell-mediated chromosome transfer.
Chimeric mice were prepared from the mouse recombinant ES cells obtained, and the CYP3A-HAC vector was transmitted from the chimeric mice to their offspring to prepare humanized CYP3A mice. Mice with these artificial chromosomes have one more chromosome than wild-type mice but normally develop and transmit the artificial chromosomes to their descendants.

Features of humanized CYP3A mouse

 

Since our CYP3A mouse contains the control region existing upstream of the human CYP3A gene, tissue-specific/time-specific expression and the expression level of the CYP3A gene are similar to humans. Since gene expression is controlled by the physiological environment as described above, our CYP3A mouse can be used for pharmacokinetic tests and toxicity tests at the individual animal level, and toxicity tests such as teratogenicity tests.

Gene expression analysis in each tissue

vector_8

mRNA was prepared from each tissue of humanized CYP3A mice and the expression specificity of CYP3A4, 3A5, and 3A43 was examined by RT-PCR. By gene expression with the human CYP3A promoter, it was shown that each CYP3A gene is expressed tissue-specifically.
down_arrow_mark The above suggests that the influence of drug candidate compounds can be effectively examined.

Time-specific expression of CYP3A in the liver


tech_02_04_image

mRNA from the liver was prepared at each stage from fetus to adult and the expression of CYP3A4 and CYP3A7 was examined by RT-PCR. As a result, CYP3A7 was expressed specifically in the fetal stage as in the case of humans, and it became clear that CYP3A4 is expressed after birth.
down_arrow_markBased on the above, it is expected that CYP3A mice can be used for teratogenicity tests of drug candidate compounds.

Expression level of CYP3A in liver microsomes (Western blot analysis)

The expression level of CYP3A in the liver of our CYP3A mouse was confirmed by Western plotting method and it was found that it is the average degree of expression of CYP3A in commercially available human liver microsome.Expression of CYP3A is greatly influenced by the living environment and it is known that individual differences are large, but expression of CYP3A in liver in our CYP3A mouse is small individual difference,it is thought to be useful as a pharmacokinetic test and a safety test in the drug discovery process.

vector_9

In vitro micronucleus assay

The in vitro micronucleus assay is a test method to measure the frequency of chromosomal aberration-inducing activities mainly with bone marrow cells for a mutagenicity candidate compound that was positive in the Ames test. An in vitro micronucleus assay using aminoanthracene (2AA) as a mutagen was carried out with S9 fraction prepared from a humanized CYP3A mouse, and as a result, in the CYP3A enzyme system, a higher micronucleus appearance rate was observed compared to when the S9 fraction derived from a mouse or a rat was used.

As described above, it is expected that more accurate analysis results can be obtained in mutagenicity tests by combining a humanized CYP3A mouse with chromosome analysis technology. The in vitro micronucleus test using primary cultured hepatocytes prepared from humanized CYP3A mice is also available, so please inquire.
vector_10

Pharmacokinetic study

We provided pharmacokinetic study contract services (non-GLP test) with humanized CYP3A mice.

Treatment of muscular dystrophy model mouse

 

The human dystrophin gene is 2.4 Mb in length and is a huge gene consisting of 79 exons.
Professor Oshimura et al. in Tottori University Graduate School of Medicine succeeded in reducing symptoms of muscular dystrophy model mice by transfecting HAC vectors carrying the human dystrophin gene into cells of muscular dystrophy model mice and performing cell transplantation.
【PubID:21849666

Artificial chromosome vector containing human dystrophin gene

r_d_02_01_image

Transplantation of cells carrying artificial chromosome vectors loaded with human dystrophin genes

r_d_02_02_image

  1. Mesodermal angioblasts (mdxMABs) are collected from a muscular dystrophy model mouse (mdx)
  2. Artificial chromosome vectors loaded with the human dystrophin gene (DYS-HAC) are transfected into mesodermal angioblasts by the microcell-mediated chromosome transfer (MMCT) method.
  3. Cells containing the DYS-HAC vector were selected and infected with lentivirus expressing MyoD and nLacZ.
  4. Evaluation in vitro (clone selection, proliferation analysis, differentiation analysis).
  5. Mesodermal angioblasts selected in Step 4 are transplanted to immunodeficient muscular dystrophy model mice (SCID/mdx).
  6. Dystrophin gene expression analysis, morphological analysis, and a function recovery test are performed.

Expression analysis in mice transplanted with cells transfected with DYS-HAC vector

r_d_02_03_image
Expression of the dystrophin protein (Dys 427 kDa) in the tibialis anterior muscle was examined by Western blotting.
In normal mice, expression of the dystrophin protein (Dys) was observed (right: Untreated SCID). On the other hand, the expression of the Dys protein was not observed in muscular dystrophy model mice (center: Untreated SCID/mdx). Mice with the Dys-HAC vector (left: Treated SCID/mdx) showed that the transfected human dystrophin gene was expressed.

Expression of FVIII gene

 

The FVIII protein is one of the blood coagulation factors, and it is known that a malfunction in this gene is the cause of hemophilia A. Professor Oshimura et al. in Tottori University succeeded in transfecting the HAC vector with tandemly linked 16 copies of the FVIII gene into CHO cells and human mesenchymal stem cells.【PubID:21833006
Expression by an artificial chromosome vector is stable for a long period of time, and it can be expected to be used for protein production systems in addition to gene therapy.

Method of loading FVIII gene into HAC vector

r_d_03_01_image
One to sixteen copies of the FVIII gene were loaded into the HAC vector.

Effect of FVIII gene copy number on production of FVIII proteins

r_d_03_02_image
The effect of the copy number of the loaded FVIII gene on the amount of FVIII protein production was evaluated. As the number of FVIII genes loaded increased, the production of FVIII proteins increased, and it was revealed that in the case of 16 copies, the amount of production was about ten times as large as in the case of 1 copy.

Long-term stability of expression

r_d_03_03_image
The expression stability of the FVIII gene in human mesenchymal stem cells was evaluated. In the system in which 16 copies of the FVIII gene were introduced into the PAC vector (◇), the expression level decreased to 10% to 40% after 50 PDLs, but the system with 16 copies of the FVIII gene on the HAC vector (□) showed little change in expression even after 50 PDLs.