Patent Description:
The fact that cell-free fetal DNA (cf-fDNA) is present in maternal blood, first proven in <NUM>, has opened a new chapter in prenatal genetic diagnosis.

In recent years, with the discovery of cell-free fetal DNA in maternal blood and the development of high-throughput sequencing technology, a new non-invasive prenatal screening technology based on sequencing free DNA in plasma of a pregnant woman has been applied in the clinical field, which can detect common chromosome aneuploidies in a fetus in a more accurate and effective way than the traditional serological screening method.

So far, cf-fDNA has been widely applied in non-invasive prenatal detection, including determination of fetal sex, autosomal genetic disease (such as β thalassemia, achondroplasia), RhD blood type and other genetic disease (including aneuploidy such as trisomy <NUM> syndrome, sex chromosome-linked disease such as X-linked hemophilia and fragile X syndrome, and the like).

Current research indicates that the cell-free DNA (cfDNA) in plasma of a pregnant woman is an extracellular small-fragment DNA which is free in blood circulation of the pregnant woman, mainly originates from apoptotic placental chorionic cells and enters the maternal blood circulation via penetrating through the placental barrier.

The biological basis of this non-invasive prenatal detection technique lies in that genomic DNA originating from the placental trophoblast cells during pregnancy is degraded into a DNA fragment of about <NUM> bp, which enters the maternal blood circulation through the maternal-fetal interface and thus can be detected in the peripheral blood, with a role similar to chorionic villus sampling. However, it is still unclear how the genomic DNA of the placental trophoblast cell is degraded into DNA fragments, by which mechanism the DNA fragment is released into the maternal circulation, and whether the degraded DNA fragment exhibits regulatory effect on the pregnancy process. Besides, the total cell-free DNA in peripheral blood of a pregnant woman is a mixture of maternal-derived and fetal-derived free DNAs, with the fetal-derived free DNA only accounting for about <NUM>% of the total free DNA on average (even lower in early pregnancy), despite its content increasing with the gestational weeks. At present, the maternal-derived free DNA and the fetal-derived free DNA cannot be distinguished by most of the high-throughput sequencing methods due to genetic similarity, which causes great technical difficulty for accurate non-invasive prenatal detection.

For example, the existing method for detecting chromosomal aneuploidy based on cell-free DNA in plasma has problems of <NUM>) an unclear source for the cell-free DNA in plasma, and <NUM>) a low concentration of fetal-derived free DNA in plasma (resulting in the fact that the aneuploidy in the plasma fetal-derived free DNA is undetectable for a sample in early pregnancy) and a large difference in samples.

Therefore, there is an urgent need in the art to develop a new method of non-invasive prenatal diagnosis with high accuracy and sensitivity.

An object of the present disclosure is to provide a method for non-invasive prenatal diagnosis with high accuracy and sensitivity.

The present invention provides a method for detecting fetal-derived DNA in a blood sample, comprising:.

In a preferred embodiment, the isolating step further comprises:.

In a further preferred embodiment, the blood sample is selected from the group consisting of plasma, serum and a combination thereof.

In a further preferred embodiment, the blood sample is a pretreated blood sample.

In a further preferred embodiment, the blood sample is a supernatant after centrifugation.

In a still further preferred embodiment, the supernatant is obtained by two steps:.

In a still further preferred embodiment, the isolating of the exosomes is conducted by using a magnetic-bead separation method, an affinity separation method or a combination thereof.

In a further preferred embodiment, the isolating is conducted by sorting or capturing with a specific antibody against at least one of the antigens PLAP, CD9, CD63 and CD81.

In a further preferred embodiment, a magnetic-bead separation method is conducted by isolating with a magnetic bead conjugated with a CD9 antibody or a PLAPI antibody on the surface.

In a further preferred embodiment, the detecting includes determining the number and/or sequence information of fetal-derived Y chromosomes.

In a still further preferred embodiment, the detecting includes determining the number and/or sequence information of fetal-derived X chromosomes.

In a further preferred embodiment, the detecting includes determining the number and/or sequence information of fetal-derived autosomes.

In a further preferred embodiment, the detecting includes determining the fetal-derived DNA fraction.

A method of the invention may further comprise for constructing a sequencing librar for the exosomal DNA to obtain an exosomal DNA sequencing library.

An exosomal DNA sequencing library thus obtained may be subjected to high-throughput sequencing to obtain information of the exosomal DNA wherein the exosomal DNA comprises fetal-derived DNA.

In a further aspect of the invention, there is provided a method for non-invasive prenatal gene detection, comprising:.

The gene detection may include determination of fetal-derived DNA fraction, determination of fetal sex, detection of autosomal genetic disease, detection of sex chromosome-linked disease, determination of RhD blood type or detection of chromosome aneuploidy.

The detection of chromosomal aneuploidy may include detection of trisomy <NUM>, trisomy <NUM> or trisomy <NUM>.

The present disclosure is provided by the present inventors based on the following findings: the present inventors, after extensive and intensive researches, unexpectedly discovered for the first time that exosomes in plasma of a pregnant woman during pregnancy comprise DNA fragments and that such exosomal DNA contains quite rich fetal-derived DNA, thus suggesting that accurate and specific information useful in prenatal diagnosis can be provided through isolating or enriching the fetal-derived DNA or total exosomal DNA containing the fetal-derived DNA. Specifically, determination of fetal-derived DNA fraction, fetal sex, autosomal genetic disease, sex chromosome-linked disease, RhD blood type and chromosomal aneuploidy can be provided accurately via high-throughput sequencing the fetal-derived DNA present in the total exosomes during pregnancy, and whole genomic sequence information can be further provided.

As used herein, the term "exosome" or "exosomes" refers to small vesicles with a diameter of about <NUM> to120 nm and a density of about <NUM> to <NUM>/ml secreted by late endosomes in living cells, which contain endosomal protein markers (such as TSG101, CD9, CD63, CD81 and the like) on the surface of lipid membrane. The exosomes are commonly present in body fluid and can be found in blood, urine, amniotic fluid and malignant tumor-caused ascites.

The change of exosome content during pregnancy is related to gestational weeks, and the exosome content in peripheral blood of a normal pregnant woman during pregnancy is significantly increased by about <NUM> to <NUM> times compared with a non-pregnant woman, with increase of the exosome content with the gestational weeks. The fetal-derived exosomes in peripheral blood of a pregnant woman are detectable in gestational weeks <NUM> to <NUM> through quantification of PLAP on the surface of the fetal-derived exosomes, and their presence increases rapidly with the gestational weeks, suggesting an increased release of placental-specific exosomes into maternal circulation with maturation of the placenta and active maternal-fetal exchange. Currently, exosomes containing fetal-derived DNA have not been found by scientific researchers.

The present disclosure is provided by the present inventors based on the findings: some exosomes are released by human placental trophoblast cells and enter the maternal circulation during pregnancy, and parts of the exosomes contain fetal-derived DNA which is useful in prenatal diagnosis of the fetus, for example as a molecular marker for chromosomal abnormality. It is indicated by the research of the present disclosure that fetal-derived DNA contained in exosomes during pregnancy can be used for detection of DNA which covers the whole genome.

Due to the similarity of fetal-derived DNA contained in exosomes and cell-free DNA (cfDNA) in plasma, the fetal-derived DNA contained in exosomes can be used as a marker for fetal DNA molecules which is similar to the plasma cfDNA. Further, the placental-specific exosome may become a new candidate entity for studying disease occurrence, development, early prediction, pathological classification, prognosis and therapeutic target during pregnancy, demonstrating a favorable application prospect.

In a specific embodiment of the present disclosure, it has been found for the first time that fetal sex and chromosomal aneuploidy can be accurately determined by high-throughput sequencing fetal-derived DNA which is present in exosomes during pregnancy. In one typical method, exosomes are isolated by combination of SBI Exoquick with CD9/PLAP immunomagnetic beads (ThermoFisher), the exosomal DNA is extracted for high-throughput sequencing, so as to detect fetal sex and aneuploidy (such as trisomy <NUM>), in which the exosomal DNA contains plenty of fetal-derived DNAs.

As used herein, the terms "capture agent" and "sorting agent" are used interchangeably and refer to agents that can capture, enrich or sort exosomes from a blood sample or a body fluid sample.

A typical capture agent includes: (a) a specific antibody against at least one of the antigens PLAP, CD9, CD63 and CD81; or (b) magnetic beads conjugated with the antibody. For example, Exoquick solution and/or immunomagnetic beads capable of specifically recognizing CD9 and/or placental alkaline phosphatase (i.e. PLAP) can be employed.

The immunomagnetic beads are spherical magnetic particles which are conjugated with monoclonal antibodies on their surfaces, and specifically capture total exosomes or fetal-derived exosomes in plasma through the interaction between the monoclonal antibody and antigens (such as CD9, CD63, CD81 and PLAP) located on the surfaces of exosomes.

Total exosomes can be isolated by methods including a precipitation and centrifugation method (SBI company), and immunomagnetic beads separation (Thermo Fisher company), which can be combined together with the advantages of obtaining a great number of exosomes by using the reagent from SBI, and overcoming the deficiency of low purity (for the exosomes obtained by SBI company) through the immunomagnetic beads separation.

After isolation, the exosomal DNA is extracted for routine library construction and high-throughput sequencing. The fetal sex and fetal-derived DNA fraction are determined according to the number and/or sequences information of Y chromosomes. The detection of RhD blood type, autosomal genetic disease, sex chromosome-linked disease and chromosome aneuploidy was conducted by statistical analysis according to the sequences information of each chromosome.

It should be noted that the total exosomal DNA extracted from peripheral blood of a pregnant woman includes both maternal-derived DNA and fetal-derived DNA. "Fetal-derived DNA fraction" as used herein refers to the ratio of the number of fetal-derived DNA molecules to the number of total exosomal DNA molecules.

In order to understand the present disclosure easily, the mechanism is provided as below for reference. It should be appreciated that the protection scope of the present disclosure is not limited by the mechanism.

The present inventors propose a scientific mechanism and explanation as below. Exosomes released from placental trophoblast cells are found to be present in maternal blood during pregnancy, which contain cell-free fetal DNA and this is more suitable as a molecular marker for detection of fetal chromosomal abnormality through high-throughput sequencing compared to plasma cell-free DNA derived from both mother and fetus.

Specifically, maternal-derived and fetal-derived exosomes are present in maternal blood during pregnancy, while exosomes derived from lymphocytes can be extracted from peripheral blood of a non-pregnant woman by chromatographic analysis and immunoadsorption. Further, not only maternal-derived exosomes but also fetal-derived exosomes have been detected in peripheral blood of a pregnant woman through quantifying with a placental-specific PLAP antibody. Thus, it has been demonstrated that the exosomes present in maternal blood during pregnancy are derived from both mother and fetus, which is similar to the plasma cell-free DNA derived from both mother and fetus in a pregnant woman.

It has been proved by in vitro and in vivo experiments that the fetal-derived exosomes in maternal blood are mainly generated in placental trophoblast cells and then released into maternal blood. In the placental trophoblast cells, primary endosomes develop into mature endosomes through invagination of cell membranes, subsequently some mature endosomes enter lysosomes but some endosomes form exosomes along with plenty of signal molecules (such as miRNA, protein and the like) via encapsulation which are subsequently released into the extracellular matrix through membrane fusion and then into maternal blood.

Despite unclear biosynthesis, transportation, inclusion encapsulation and efficacy of exosome, it is suggested by the present findings that fetal-derived exosomes can be involved in regulation of some important processes during pregnancy, such as immune tolerance, maternal-fetal interface remodeling, inflammatory response and the like by actively phagocytizing tissue-specific inclusions and releasing them into the maternal circulation. In addition, the exosomes have a stable bilayer lipid membrane, which seems to be more helpful in maintaining the stability of fetal-derived DNA.

The present disclosure provides a method for detecting fetal DNA based on exosomes in peripheral blood of a pregnant woman, where the method may be diagnostic and may be non-diagnostic.

Typically, a method of the present disclosure includes the steps of:.

In a method of the present disclosure, peripheral blood of a pregnant woman can be collected by a conventional method, and then plasma or serum in the peripheral blood is separated. For example, the peripheral blood (e.g., in a volume of about <NUM> to <NUM>, more preferably <NUM> to <NUM>) is collected by using a commercially available Streck tube. Plasma or serum can be separated by using a two-step method.

From the separated plasma or serum, the total exosomes can be isolated by centrifugation or the like. Preferably, the exosomes which contain fetal-derived DNA, among the total exosomes obtained, may be further enriched or captured, for example, with magnetic beads conjugated with CD9/PLAP antibody through the magnetic-beads separation method.

From the isolated total exosomes or fetal-derived exosomes, DNA in the exosomes may be extracted, thus fetal-derived DNA can be detected. For example, firstly the fetal-derived DNA can be subjected to high-throughput sequencing library construction, followed by sequencing and analyzing, so as to detect fetal sex, Rh blood type, chromosome aneuploidy, genetic abnormality and the like.

In another preferred embodiment, a typical method of the present disclosure includes steps as below.

Step <NUM>: Plasma exosomes are isolated from a blood sample with known methods or reagents, such as the commercially available SBI quick reagent from SBI Company.

Step <NUM>: Fetal-derived plasma exosomes are enriched or captured from the plasma exosomes obtained in step <NUM>. For example, after dissolving in a PBS buffer, the exosome precipitate generated in step <NUM> is captured or enriched with CD9/PLAP immunomagnetic beads, followed by incubating at a temperature (e.g., <NUM> to <NUM>) for a time period (e.g., <NUM> to <NUM> hours, or overnight), thus obtaining a magnetic bead-exosome binary complex. The CD9/PLAP immunomagnetic beads can be prepared by conventional methods or are commercially available (for example, CD9/PLAP immunomagnetic beads purchased from Thermo Fisher).

Step <NUM>: DNA is extracted from the magnetic bead-exosome binary complex obtained in step <NUM>, followed by library construction (with increased PCR cycles) and/or sequencing, so as to obtain sequencing data.

Step <NUM>: The sequencing data is subjected to information analysis, thus obtaining corresponding analysis results, including for example the fetal-derived DNA fraction, fetal sex, RhD blood type, presence or absence of chromosomal aneuploidy, autosomal genetic disease and sex chromosome-linked disease and the like.

The present disclosure is further illustrated as below in combination with specific examples. It is to be understood that the examples are not intended to limit the scope of the disclosure. Experimental methods in the examples in which the specific conditions are not indicated are generally conducted in accordance with conventional conditions, for example, the conditions described in <NPL>) or in accordance with the conditions instructed by a manufacturer. Percentage and parts are by weight unless otherwise stated.

Samples were plasma samples derived from <NUM> normal males, <NUM> pregnant women with a normal male fetus, <NUM> pregnant women with a normal female fetus, <NUM> pregnant women with a fetus suffering from trisomy <NUM> (two male fetuses and two female fetuses respectively) and <NUM> pregnant women with a fetus suffering from trisomy <NUM> (one male fetus and one female fetus) which were obtained by the step of plasma separation in <NUM>. <NUM>, in which the information on fetal sex and chromosomal aneuploidy of the plasma samples had been acquired by existing detection techniques.

Exosomal DNA was extracted by using the Magen MagPure Circulating DNA Mini KF Kit.

Table <NUM> shows the detection results, which indicate correct detection of fetal sex for all samples, correct detection of trisomy <NUM> for <NUM> samples and correct detection of trisomy <NUM> for <NUM> samples. The results obtained are identical to the information of corresponding plasma samples which had been acquired by existing detection techniques.

The plasma was separated by the method in <NUM>.

The fetal-derived exosomes in plasma were isolated by the steps in <NUM>. <NUM> except that the magnetic beads conjugated with CD9 antibody were replaced with magnetic beads conjugated with PLAP antibody.

Table <NUM> shows the detection results, which indicate correct detection of fetal sex for all samples, with results identical to those detected with plasma cell-free DNA (cfDNA) by the existing method for fetal sex determination.

The total plasma exosomes isolated with ExoQuick Exosome Precipitation Solution in Example <NUM> and the exosomes isolated with CD9/PLAP magnetic beads were assayed with the Western Blot method, with results shown in <FIG>.

<FIG> shows the results, which indicate the total plasma exosomes isolated with the ExoQuick Exosome Precipitation Solution and the exosomes isolated with CD9/PLAP magnetic beads both contain plenty of PLAP proteins, suggesting successful isolation of fetal-derived exosomes.

Claim 1:
A method for detecting fetal-derived DNA in a blood sample, comprising:
(i) subjecting a blood sample from peripheral blood of a pregnant woman to an isolating step, so as to obtain the exosomal DNA,
wherein the exosomal DNA comprises fetal-derived DNA and
(ii) detecting the presence of the fetal-derived DNA in the exosomal DNA,
wherein the presence of the fetal-derived DNA in the exosomal DNA is detected by at least one technique selected from PCR amplification, sequence-specific probe capturing technology and high-throughput sequencing technology.