CN114807338A

CN114807338A - HLA typing method and system

Info

Publication number: CN114807338A
Application number: CN202110111896.0A
Authority: CN
Inventors: 王志敏; 虞修简; 黄薇; 郑华军
Original assignee: SHANGHAI INDUSTRIAL TECHNOLOGY INSTITUTE; Chinese National Human Genome Center at Shanghai
Current assignee: SHANGHAI INDUSTRIAL TECHNOLOGY INSTITUTE; Chinese National Human Genome Center at Shanghai
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2022-07-29

Abstract

The invention relates to a method and a system for carrying out HLA typing on iPS cells, which comprises the step of carrying out HLA gene detection on the iPS cells and differentiated cells thereof. The invention also relates to reagents used in the method.

Description

HLA typing method and system

Technical Field

The invention relates to the field of molecular biology, in particular to a method and a system for carrying out HLA typing on iPS cells and differentiated cells thereof.

Background

Human leukocyte antigen, i.e. hla (humanleukocyteatigen), is the major genetic system regulating the human body-specific immune response and determining individual differences in disease susceptibility, and is closely associated with rejection in allogeneic organ transplantation.

The HLA system plays an important role in antigen recognition, antigen presentation, immune response and regulation, destruction of foreign antigen target cells and the like, and is the main material basis for causing immunological rejection. Both class I and class II antigens on the cell surface of the graft are strong graft antigens, and both humoral and cellular immunity are involved in rejection of the graft, and it is critical that HLA matching between recipients be successful, whether allogeneic organ, tissue or cell transplantation.

The construction of a human iPS library covering HLA typing of a large proportion of the population is an important link to the upstream construction of stem cell therapy. The HLA typing of a sample can be obtained by sequencing the genome. The phenomenon of inaccurate typing of the human iPS library obtained in the prior art still exists, the typing result is influenced, and immune rejection reaction can be possibly caused. There remains a need in the art for improved methods of HLA genotyping iPS cells in order to accurately type HLA genes.

Disclosure of Invention

The inventors found that HLA sequencing only on somatic cells and iPS cells and using them as a basis for typing may present a hidden risk.

In a first aspect, the invention provides a primer for amplifying mammalian cell DNA, the amplification product of the primer comprising one or more fragments selected from the group consisting of:

fragments of HLA-A gene amplified by using SEQ ID NO 1 and 2 as primers,

fragments of HLA-B gene amplified by using SEQ ID NO. 3 and 4 as primers,

fragments of HLA-C gene amplified by using SEQ ID NO 5 and 6 as primers,

fragments of HLA-DPA gene amplified by using SEQ ID NOS 7 and 8 as primers,

fragments of HLA-DPB gene amplified by using SEQ ID NO 9 and 10 as primers,

the HLA-DQA gene fragment amplified by using SEQ ID NO 11 and 12 as primers,

the HLA-DQB gene fragment amplified by using SEQ ID NO 13 and 14 as primers,

fragments of HLA-DRB gene amplified by using SEQ ID NO:15 and 16 as primers.

In one or more embodiments, the primer is a sequence that hybridizes under stringent conditions to one or more fragments selected from the group consisting of:

fragments of HLA-A gene amplified by using SEQ ID NO 1 and 2 as primers,

fragments of HLA-B gene amplified by using SEQ ID NO. 3 and 4 as primers,

fragments of HLA-C gene amplified by using SEQ ID NO 5 and 6 as primers,

fragments of HLA-DPA gene amplified by using SEQ ID NOS 7 and 8 as primers,

fragments of HLA-DPB gene amplified by using SEQ ID NO 9 and 10 as primers,

the HLA-DQA gene fragment amplified by using SEQ ID NO 11 and 12 as primers,

the HLA-DQB gene fragment amplified by using SEQ ID NO 13 and 14 as primers,

fragments of HLA-DRB gene amplified by using SEQ ID NO:15 and 16 as primers.

In one or more embodiments, the primer is selected from the group consisting of:

(1) 1-16 of any of the sequences shown in SEQ ID NOs,

(2) a sequence having at least 90% identity to (1); and

(3) a mixture of the sequences of (1) and (2).

(1) a primer pair selected from SEQ ID NO 1 and SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14, and/or SEQ ID NO 15 and SEQ ID NO 16,

(2) a sequence having at least 90% identity to (1); and

(3) a mixture of the sequences of (1) and (2).

In one or more embodiments, the mammal is a human.

In one or more embodiments, the cell is an iPS cell or a differentiated cell thereof.

In one or more embodiments, the DNA is genomic DNA.

The present invention also provides a probe for detecting HLA genes in mammalian cells, comprising:

(1) a probe that recognizes one or more fragments selected from the group consisting of:

fragments of HLA-A gene amplified by using SEQ ID NO 1 and 2 as primers,

fragments of HLA-B gene amplified by using SEQ ID NO. 3 and 4 as primers,

fragments of HLA-C gene amplified by using SEQ ID NO 5 and 6 as primers,

fragments of HLA-DPA gene amplified by using SEQ ID NOS 7 and 8 as primers,

fragments of HLA-DPB gene amplified by using SEQ ID NO 9 and 10 as primers,

the HLA-DQA gene fragment amplified by using SEQ ID NO 11 and 12 as primers,

the HLA-DQB gene fragment amplified by using SEQ ID NO 13 and 14 as primers,

fragments of HLA-DRB gene amplified by using SEQ ID NO 15 and 16 as primers, and/or

(2) The complementary sequence of (1).

In one or more embodiments, the mammal is a human.

The present invention also provides a kit for detecting HLA genes in mammalian cells, which comprises

(1) Primers for detecting HLA gene, and

optionally (2) a probe for detecting an HLA gene,

wherein the HLA gene is selected from one or more of the group consisting of: HLA-A, HLA-B, HLA-C, HLA-DPA, HLA-DPB, HLA-DQA, HLA-DQB, and HLA-DRB.

In one or more embodiments, the HLA genes are selected from one or more of the following groups: (a) HLA-A, HLA-B, HLA-C, HLA-DPA, HLA-DQA, HLA-DQB, and (b) HLA-C, HLA-DPB.

In one or more embodiments, the kit is for HLA typing a differentiated cell of an iPS cell or reducing the risk of matching a differentiated cell of an iPS cell.

In one or more embodiments, the kit comprises the primers described in any of the embodiments herein, optionally the probes described in any of the embodiments herein, and optionally reagents required for PCR.

In one or more embodiments, the mammal is a human.

A second aspect of the present invention relates to a method for HLA typing or reducing the risk of matching a differentiated cell of an iPS cell, comprising performing one or more HLA gene tests on the differentiated cell of the iPS cell, and determining the genotype of the differentiated cell of the iPS cell based on the results of the HLA gene tests.

In one or more embodiments, the method can further comprise:

optionally (1) detecting one or more HLA genes of the iPS cell,

(2) detecting the one or more HLA genes of a differentiated cell of the iPS cell,

and optionally (3) comparing the detection results of the HLA genes of the iPS cell and the differentiated cell thereof, and determining the HLA typing result of the iPS cell when the detection results are the same.

In one or more embodiments, the iPS cell is a human iPS cell.

In one or more embodiments, the iPS cell is SHI001 or DYR.

In one or more embodiments, the differentiated cell is a cardiomyocyte.

In one or more embodiments, the detection of the HLA gene is performed by PCR amplification.

In one or more embodiments, the HLA gene is selected from one or more of the following: HLA-A, HLA-B, HLA-C, HLA-DPA, HLA-DPB, HLA-DQA, HLA-DQB, and HLA-DRB.

In one or more embodiments, the detection of the HLA gene is the size and/or number of amplified fragments.

In one or more embodiments, the HLA gene is detected as a number of amplified fragments comprising a nucleic acid sequence selected from one or more of:

an about 4.8kb nucleic acid sequence of HLA-A gene amplified by using SEQ ID NOS: 1 and 2 as primers,

an about 4.3kb nucleic acid sequence of HLA-B gene amplified by using SEQ ID NOS.3 and 4 as primers,

an about 5.8kb nucleic acid sequence of HLA-C gene amplified by using SEQ ID NOS 5 and 6 as primers,

about 9.8k nucleic acid sequence of HLA-DPA gene amplified by using SEQ ID NOS: 7 and 8 as primers,

about 9.1kb of the nucleic acid sequence of HLA-DPB gene amplified from SEQ ID NOS: 9 and 10 as primers,

the about 7.3kb nucleic acid sequence of HLA-DQA gene amplified by using SEQ ID NO 11 and 12 as primers,

13 and 14 as primers, the HLA-DQB gene of about 8.2kb nucleic acid sequence,

about 9.8kb of the nucleic acid sequence of HLA-DRB gene amplified by using SEQ ID NOS: 15 and 16 as primers.

In one or more embodiments, the PCR is performed using primers as described in any of the embodiments of the first aspect herein.

In one or more embodiments, the PCR is performed using a primer pair selected from SEQ ID NO 1 and 2, SEQ ID NO 3 and 4, SEQ ID NO 5 and 6, SEQ ID NO 7 and 8, SEQ ID NO 9 and 10, SEQ ID NO 11 and 12, SEQ ID NO 13 and 14, and/or SEQ ID NO 15 and 16.

The invention also provides the use of a primer according to any embodiment herein and optionally a probe according to any embodiment herein in the preparation of a kit for HLA typing a differentiated cell of an iPS cell or reducing the risk of matching a differentiated cell of an iPS cell.

In one or more embodiments, the HLA typing or typing risk reducing step comprises performing one or more HLA gene tests on the differentiated cells of the iPS cells, and determining the genotype of the differentiated cells of the iPS cells based on the results of the HLA gene tests.

In one or more embodiments, the HLA-typing or typing risk reducing step comprises:

optionally (1) detecting one or more HLA genes of the iPS cell,

In one or more embodiments, the iPS cell is a human iPS cell.

In one or more embodiments, the iPS cell is SHI001 or DYR.

In one or more embodiments, the differentiated cell is a cardiomyocyte.

In one or more embodiments, the primers are used for detection of the HLA gene by PCR amplification.

13 and 14 as primers, the HLA-DQB gene of about 8.2kb nucleic acid sequence,

The invention also provides the use of a primer according to any embodiment herein and optionally a probe according to any embodiment herein in the preparation of a medicament comprising differentiated cells of iPS cells.

In one or more embodiments, the medicament is for treating a disease that benefits from differentiated cells of the iPS cells. Preferably, the disease benefiting from the differentiated cells of the iPS cells is a disease benefiting from the transplantation of differentiated cells of iPS cells, such as a disease requiring the transplantation of the differentiated cells.

In one or more embodiments, the primers and/or probes are used to HLA-type or reduce the risk of typing differentiated cells of iPS cells.

In one or more embodiments, the HLA typing or reduced risk of matching step comprises performing one or more HLA gene tests on the differentiated cell of the iPS cell, and determining the genotype of the differentiated cell of the iPS cell based on the results of the HLA gene tests.

optionally (1) detecting one or more HLA genes of the iPS cell,

In one or more embodiments, the iPS cell is a human iPS cell.

In one or more embodiments, the iPS cell is SHI001 or DYR.

In one or more embodiments, the differentiated cell is a cardiomyocyte.

13 and 14 as primers, the HLA-DQB gene of about 8.2kb nucleic acid sequence,

The third aspect of the present invention relates to a method for amplifying an HLA gene fragment of a sample, the HLA gene being selected from one or more of: HLA-A, HLA-B, HLA-C, HLA-DPA, HLA-DPB, HLA-DQA, HLA-DQB, and HLA-DRB.

In one or more embodiments, the sample is an iPS cell and/or differentiated cells thereof.

In one or more embodiments, the iPS cell is a human iPS cell.

In one or more embodiments, the iPS cell is SHI001 or DYR.

In one or more embodiments, the differentiated cell is a cardiomyocyte.

In one or more embodiments, the amplification is performed by PCR amplification.

A fourth aspect of the present invention provides an apparatus for HLA typing a differentiated cell of an iPS cell or reducing the risk of matching a differentiated cell of an iPS cell, the apparatus comprising a memory, a processor and a computer program stored on the memory and executable on the processor, wherein the processor when executing the program implements the steps of:

(1) detecting the one or more HLA genes of a differentiated cell of the iPS cell,

(2) and determining the genotype of the differentiated cell of the iPS cell according to the result of the HLA gene detection.

In one or more embodiments, the steps include:

optionally (1) detecting one or more HLA genes of the iPS cell,

In one or more embodiments, the iPS cell is a human iPS cell.

In one or more embodiments, the iPS cell is SHI001 or DYR.

In one or more embodiments, the differentiated cell is a cardiomyocyte.

about 9.1kb of the nucleic acid sequence of HLA-DPB gene amplified by using SEQ ID NOS.9 and 10 as primers,

about 8.2kb of the nucleic acid sequence of HLA-DQB gene amplified by using SEQ ID NO. 13 and 14 as primers,

In one or more embodiments, the other features of the device are as described herein for the method of the second aspect.

Detailed Description

The inventors found that HLA sequencing only on somatic cells and iPS cells and using them as a basis for typing may present a hidden risk. The iPS cell may also have HLA gene change in the differentiation process, which affects the matching result and may cause immune rejection.

Therefore, the inventors have found through intensive studies that HLA-A, HLA-B, HLA-C, HLA-DPA, HLA-DPB, HLA-DQA, HLA-DQB, and HLA-DRB of iPS cells and differentiated cells thereof can be accurately HLA-typed. In some embodiments, the HLA genes to be detected are: (a) HLA-A, HLA-B, HLA-C, HLA-DPA, HLA-DQA, HLA-DQB, or (b) HLA-C, HLA-DPB.

A "sample" as described herein is any type of polynucleotide-containing sample from a subject. Preferably, the sample described herein is an established iPS cell and/or differentiated cells thereof. Alternatively, iPS cells can be reprogrammed to establish from somatic cells. The process of establishing iPS cells mainly comprises the following steps: (1) isolating and culturing the host cell; (2) introduction of pluripotency-related genes into host cells by virus-mediated or other means (e.g., non-integrative (episomal) plasmids); (3) planting the virus infected cells on feeder layer cells, culturing in a special culture system for ES cells, and adding corresponding small molecular substances to promote reprogramming according to requirements during culture; (4) identification of iPS cells was performed after the occurrence of ES-like clones. Herein, an iPS cell is typically a human iPS cell, such as SHI001 or DYR.

Differentiated cells include, but are not limited to, cardiomyocytes.

The term "nucleic acid" or "polynucleotide" refers to a Deoxyribonucleotide (DNA) or ribonucleotide polymer (RNA) in either single-or double-stranded form, and the complement thereof. Nucleic acids contain synthetic, non-natural or altered nucleotide bases. The nucleotide may be a ribonucleotide, a deoxyribonucleotide, or a modified form thereof. Examples of polynucleotides contemplated herein include single-and double-stranded DNA, single-and double-stranded RNA, and hybrid molecules having a mixture of single-and double-stranded DNA and RNA. The DNA may be the coding strand or the non-coding strand. In one or more embodiments, the sample comprises fragmented genomic DNA. Methods for obtaining genomic DNA and fragmenting are well known in the art.

The basic unit of DNA is deoxyribonucleotide, which is condensed by phosphodiester bond to form a long chain molecule. Each deoxyribonucleotide consists of a phosphate, a deoxyribose, and a base. Bases (bp) of DNA are mainly adenine (A), guanine (G), cytosine (C) and thymine (T). In the double-helix structure of double-stranded DNA, A is paired with T through hydrogen bonds, and G is paired with C through hydrogen bonds. The form of DNA includes cDNA, genomic DNA, fragmented DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be of any length, for example, 10-10kbp, 20-5kbp, 30-1kbp, 50-500bp, 100-400bp, 150-300bp or 200-250 bp.

As used herein, a "primer" refers to a nucleic acid molecule having a specific nucleotide sequence that directs the synthesis at the initiation of nucleotide polymerization. The primer composition comprises one or more primers. Primers are typically two oligonucleotide sequences synthesized artificially, one complementary to one DNA template strand at one end of the target region and the other complementary to the other DNA template strand at the other end of the target region, and function as an initiation point for nucleotide polymerization. Primers designed artificially in vitro are widely used in Polymerase Chain Reaction (PCR), qPCR, sequencing, probe synthesis, and the like. The primer may be of any length, for example 5-200bp, 10-100bp, 20-800bp or 25-50 bp.

The primer of the invention is used for detecting HLA genes or fragments thereof. Illustratively, the primer: (1) can amplify the sequence represented by SEQ ID NO:1 and 2 as primers, (2) can amplify a fragment of the HLA-a gene consisting of SEQ ID NO:3 and 4 as primers, and (3) can amplify a fragment of the HLA-B gene consisting of SEQ ID NO:5 and 6 as primers, (4) can amplify a fragment of the HLA-C gene consisting of SEQ ID NO:7 and 8 as primers, (5) can amplify a fragment of the HLA-DPA gene consisting of SEQ ID NO:9 and 10 as primers, (6) can amplify a fragment of the HLA-DPB gene consisting of SEQ ID NO:11 and 12 as primers, and (7) can amplify a fragment of the HLA-DQA gene consisting of SEQ ID NO:13 and 14 as primers, amplifying the HLA-DQB gene fragment, or (8) capable of amplifying a polypeptide consisting of SEQ ID NO:15 and 16 as primers for the amplified fragment of the HLA-DRB gene.

In one or more embodiments, the primer has: (1) 1-16, (2) a sequence having at least 90% identity to (1); or (3) a mixture of the sequences of (1) and (2). In one or more embodiments, the primers are primer pairs having (1) a sequence selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, and/or SEQ ID NO. 15 and SEQ ID NO. 16, respectively, (2) a sequence having at least 90% identity to (1); and (3) mixtures of the sequences described in (1) and (2). In discussing primers, "recognition" as used herein means that the primers hybridize to the template sequence under stringent or highly stringent conditions, and that the fragments amplified by the pair of primers comprise: (1) fragments of HLA-A gene amplified by SEQ ID NOS: 1 and 2 as primers, (2) fragments of HLA-B gene amplified by SEQ ID NOS: 3 and 4 as primers, (3) fragments of HLA-C gene amplified by SEQ ID NOS: 5 and 6 as primers, (4) fragments of HLA-DPA gene amplified by SEQ ID NOS: 7 and 8 as primers, (5) fragments of HLA-DPB gene amplified by SEQ ID NOS: 9 and 10 as primers, (6) fragments of HLA-DQA gene amplified by SEQ ID NOS: 11 and 12 as primers, (7) fragments of HLA-DQB gene amplified by SEQ ID NOS: 13 and 14 as primers, or (8) fragments of HLA-DRB gene amplified by SEQ ID NOS: 15 and 16 as primers.

Stringent conditions for hybridization of the nucleic acids described herein are known to those skilled in the art. Preferably, the conditions are such that the sequences are at least about 65%, 70%, 75%, 85%, 90%, 95%, 98% or 99% homologous to each other, typically remaining hybridized to each other. Non-limiting examples of stringent hybridization conditions are hybridization in a high salt buffer containing 6XSSC, 50mM Tris-HCl (pH7.5), 1m MEDTA, 0.02% PVP, 0.02% Ficolll, 0.02% BSA and 500mg/ml denatured salmon sperm DNA at 65 ℃ and optionally washed once or twice in 0.2XSSC, 0.01% BSA at 50 ℃.

The present invention may also employ a probe to detect the HLA gene of the present invention or a fragment thereof. A "probe" as used herein is a nucleic acid sequence (DNA or RNA) that recognizes a sequence of interest (complementary to the sequence of interest). The probe is combined with the target gene through molecular hybridization to generate a hybridization signal, thereby displaying the target gene. The probe may include the entire sequence of interest or may be a fragment of the sequence of interest. The probe may be DNA or RNA transcribed therefrom. Typically, the probe carries a detectable label, such as a fluorescent label. Such fluorescent labels include, but are not limited to FAM, CY5, and VIC. Fluorescent labels suitable for use with the probes herein and methods of attaching them to the probes are known in the art.

Illustratively, the probe comprises one or more selected from the group consisting of: (1) a probe that recognizes one or more fragments selected from the group consisting of: a fragment of HLA-A gene amplified by using SEQ ID NOS: 1 and 2 as primers, a fragment of HLA-B gene amplified by using SEQ ID NOS: 3 and 4 as primers, a fragment of HLA-C gene amplified by using SEQ ID NOS: 5 and 6 as primers, a fragment of HLA-DPA gene amplified by using SEQ ID NOS: 7 and 8 as primers, a fragment of HLA-DPB gene amplified by using SEQ ID NOS: 9 and 10 as primers, a fragment of HLA-DQA gene amplified by using SEQ ID NOS: 11 and 12 as primers, a fragment of HLA-DQB gene amplified by using SEQ ID NOS: 13 and 14 as primers, a fragment of HLA-DRB gene amplified by using SEQ ID NOS: 15 and 16 as primers, and/or a complementary sequence of (2) (1).

The term "variant" or "mutant" as used herein refers to a polynucleotide that has a nucleic acid sequence altered by insertion, deletion or substitution of one or more nucleotides compared to a reference sequence, while retaining its ability to hybridize to other nucleic acids. A mutant according to any of the embodiments herein comprises a nucleotide sequence having at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to a reference sequence (SEQ ID NOS: 1-12 as described herein) and retaining the biological activity of the reference sequence. Sequence identity between two aligned sequences can be calculated using, for example, BLASTn from NCBI. Mutants also include nucleotide sequences that have one or more mutations (insertions, deletions, or substitutions) in the reference sequence and in the nucleotide sequence, while still retaining the biological activity of the reference sequence. The plurality of mutations typically refers to within 1-10, such as 1-8, 1-5, or 1-3. The substitution may be a substitution between purine nucleotides and pyrimidine nucleotides, or a substitution between purine nucleotides or between pyrimidine nucleotides. The substitution is preferably a conservative substitution. For example, conservative substitutions with nucleotides of similar or analogous properties are not typically made in the art to alter the stability and function of the polynucleotide. Conservative substitutions are, for example, exchanges between purine nucleotides (A and G), exchanges between pyrimidine nucleotides (T or U and C). Thus, substitution of one or more sites with residues from the same in the polynucleotides of the invention will not substantially affect their activity. When referring to mutants having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% sequence identity to a primer (e.g.SEQ ID NO:1-2, 4-5, 7-8) or probe according to the invention, preferably such mutants will hybridize under high stringency conditions to the corresponding DNA sequences comprising SEQ ID NO:3, 6, 9. The high stringency conditions can be hybridization and membrane washing in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃.

The present invention also provides a kit for amplifying HLA genes or HLA typing differentiated cells of iPS cells or reducing the risk of matching differentiated cells of iPS cells, comprising the primers described in any embodiment herein, optionally the probes described in any embodiment herein, and optionally reagents required for PCR. Other reagents required for PCR are known in the art, e.g., dNTPs, buffer, MgSO ₄ Polymerase, etc. Thus, the invention also includes the use of the primers described herein and optionally the probes described herein in the preparation of a kit for HLA typing a differentiated cell of an iPS cell or reducing the risk of typing of a differentiated cell of an iPS cell.

Another aspect of the present invention provides a method for HLA-typing a differentiated cell of an iPS cell or reducing the risk of matching a differentiated cell of an iPS cell, comprising performing HLA gene detection on a differentiated cell of an iPS cell and determining the genotype of the differentiated cell of the iPS cell based on the result of the HLA gene detection. The art has used the results of typing of iPS cells to indicate the typing and use (e.g., therapeutic use) of differentiated cells. By simultaneously detecting HLA genes of differentiated cells of iPS cells, the cells can be accurately subjected to HLA typing before use, and the risk caused by matching with the typing result of the iPS cells is avoided. The HLA gene is selected from one or more of: HLA-A, HLA-B, HLA-C, HLA-DPA, HLA-DPB, HLA-DQA, HLA-DQB, and HLA-DRB.

The method for detecting HLA genes is, for example, amplification of cells or DNA thereof by PCR. Other reagents than primers and/or probes required in HLA gene detection methods are known in the art. HLA typing results can be obtained by analysis of PCR amplification products. Such as gel electrophoresis, fluorescence quantitative probe method or HRM high resolution melting curve method or DNA sequencing, the procedures of these methods and the reagents used are well known in the art. DNA sequencing includes first, second and third generation sequencing.

For example, HLA typing can be determined by the size and/or number of PCR amplified fragments. Illustratively, the detection of the HLA gene is the number of amplified fragments comprising a nucleic acid sequence selected from one or more of: consisting of SEQ ID NO:1 and 2 as primers, and the sequence of about 4.8kb of the HLA-A gene amplified from SEQ ID NO:3 and 4 as primers, and the nucleic acid sequence of about 4.3kb of the HLA-B gene amplified by SEQ ID NO:5 and 6 as primers, the nucleotide sequence of about 5.8kb of HLA-C gene amplified by SEQ ID NO:7 and 8 as primers, the sequence of about 9.8k nucleic acid of HLA-DPA gene consisting of SEQ ID NO:9 and 10 as primers, the nucleic acid sequence of about 9.1kb of the HLA-DPB gene amplified from SEQ ID NO:11 and 12 as primers, the nucleic acid sequence of about 7.3kb of HLA-DQA gene amplified by SEQ ID NO:13 and 14 as primers, the nucleic acid sequence of about 8.2kb of HLA-DQB gene amplified by SEQ ID NO:15 and 16 as primers, and the amplified nucleic acid sequence of about 9.8kb of HLA-DRB gene. The PCR may be performed using the primers described herein, or may be performed using other primers whose amplification products comprise the nucleic acid sequences described above. Illustratively, the PCR is performed using a primer pair selected from SEQ ID NO 1 and 2, SEQ ID NO 3 and 4, SEQ ID NO 5 and 6, SEQ ID NO 7 and 8, SEQ ID NO 9 and 10, SEQ ID NO 11 and 12, SEQ ID NO 13 and 14, and/or SEQ ID NO 15 and 16.

Herein, the method of extracting DNA in a sample (e.g., differentiated cells) is not particularly limited, and DNA extraction methods suitable for use herein are well known in the art.

As described above, by simultaneously detecting HLA genes of differentiated cells of iPS cells, HLA typing of the cells can be accurately performed, and the risk of matching based on the typing result of the iPS cells can be avoided. Thus, the primers and/or probes described herein can be used to prepare a medicament for differentiated cells comprising iPS cells. The medicament may be used to treat diseases benefiting from the differentiated cells of the iPS cells, for example diseases benefiting from the transplantation of differentiated cells of iPS cells, including but not limited to diseases requiring the transplantation of the differentiated cells, such as heart disease, cancer. The primers and/or probes are used for HLA typing of differentiated cells of iPS cells or reducing the risk of matching of differentiated cells of iPS cells. The methods and steps for HLA typing differentiated cells of iPS cells or reducing the risk of typing differentiated cells of iPS cells are as described elsewhere herein.

The pharmaceutical compositions herein contain differentiated cells as described herein, and pharmaceutically acceptable excipients including, but not limited to, diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. Adjuvants are preferably non-toxic to recipients at the dosages and concentrations employed. Such adjuvants include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition may contain a substance for improving, maintaining or retaining, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. These substances are known from the prior art. The optimal pharmaceutical composition can be determined depending on the intended route of administration, mode of delivery and the desired dosage. Cell-containing pharmaceutical compositions for in vivo administration (e.g., transplantation) are typically provided in the form of sterile preparations. Suitable excipients for use in the pharmaceutical compositions described herein are known to those skilled in the art.

The invention also provides methods of treating a patient, particularly a patient in need of cell transplantation, by administering the cells of any of the embodiments of the invention or a pharmaceutical composition thereof. Herein, the terms "patient," "subject," "individual," "subject" are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. "treatment" refers to the subject taking a treatment regimen described herein to achieve at least one positive therapeutic effect. The treatment regimen effective to treat a patient may vary depending on factors such as the disease state, age, weight of the patient, and the ability of the therapy to elicit an immune response in the subject.

The therapeutically effective amount of a pharmaceutical composition comprising cells of the invention to be employed will depend, for example, on the degree of treatment and the goal. One skilled in the art will appreciate that the appropriate dosage level for treatment will vary depending, in part, on the cells delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and/or condition (age and general health) of the patient.

The route of administration of the cell-containing pharmaceutical compositions described herein is according to known methods, e.g., injection by intralesional route, by sustained release system or by implantation device.

The present invention also provides an apparatus for HLA typing a differentiated cell of an iPS cell or reducing the risk of matching a differentiated cell of an iPS cell, the apparatus comprising a memory, a processor and a computer program stored on the memory and executable on the processor, wherein the processor when executing the program implements the steps of: (1) detecting the one or more HLA genes of a differentiated cell of the iPS cell, (2) determining the genotype of the differentiated cell of the iPS cell according to the result of the HLA gene detection. Other features of the device are as described herein for the method of HLA-typing differentiated cells of iPS.

Furthermore, a computer-readable storage medium is disclosed, in which a computer program is stored, which computer program stored on the storage medium is operative to perform the methods described herein. The steps of a method or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by a processor, or in a combination of the two. If implemented in software as a computer program product, the functions may be stored on or transmitted over as one or more instructions or code on a computer-readable medium. Computer-readable media includes both computer storage media and communication media including any medium that facilitates transfer of a computer program from one place to another. A storage media may be any available media that can be accessed by a computer. By way of example, and not limitation, such computer-readable media can comprise RAM, ROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to carry or store desired program code in the form of instructions or data structures and that can be accessed by a computer. An exemplary storage medium is coupled to the processor such the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium may be integral to the processor. The processor and the storage medium may reside in an ASIC. The ASIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal.

According to some embodiments of the present invention, HLA typing by performing the HLA gene assay described herein on a test sample further comprises: extracting DNA in a differentiated cell and an optional iPS cell of the iPS cell; carrying out PCR of DNA using any one pair of primers SEQ ID NO 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16 to obtain amplification products; subjecting the amplification product to gel electrophoresis or DNA sequencing analysis (e.g., RSII sequencer sequencing) to obtain results for the HLA genes described herein in the DNA, e.g., genotype, fragment size, and/or fragment number; and determining the HLA type of the sample according to the result of the HLA genes, or determining the HLA genes based on the comparison according to the result of comparing the iPS cells and the differentiated cells thereof, and determining the HLA type of the sample when the detection results of the iPS cells and the differentiated cells thereof are the same.

The invention has the advantages that:

the invention utilizes a molecular biology method to detect HLA genes of iPS cells and differentiated cells thereof, and accurately carries out HLA typing on the iPS cells and the differentiated cells thereof. The risk that only the HLA sequencing result of the iPS cell and/or the somatic cell is used as the matching basis is avoided.

The present invention will be illustrated below by way of specific examples. It is to be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The materials, reagents and methods not specifically described in the examples are not conventional in the art.

Examples

Example 1

To facilitate the difference in the HLA detection results of different cells, the inventors performed HLA typing on 2 human iPS cells and their differentiated cardiomyocytes. The HLA typing of human iPS cells was found to be different from that of cardiac myocytes differentiated therefrom.

iPS cell DYR is obtained by reprogramming neonatal foreskin cells and is obtained from a Shanghai cell bank of Chinese academy of sciences; the iPS cell SHI001 was obtained by reprogramming adult fibroblasts, and was constructed by the inventors. Reprogramming step: non-integrative (episomal) plasmids are introduced into fibroblasts by an electrotransfer reagent (Lonza), cultured until the clone morphology of the pluripotent stem cells appears, and the clone of the pluripotent stem cells is selected, amplified and identified for the pluripotency. Cardiomyocyte differentiation and production conditions were as described in CN 202010288840.8.

1. HLA fragment PCR

The full-length primers are designed on the genome level for A, B, C genes of HLAI type and five genes of DPA, DPB, DQA, DQB and DRB of II type, wherein two genes of DPB and DRB are longer, and the designed primers mainly cover coding regions and have 8 fragments (the length of the fragments is 4.5-10 kb). The primers are shown in the following table.

Table 1, primers are as follows:

the PCR product sizes and conditions are shown in the following table:

TABLE 2 details of PCR

Gene	Fragment size	Annealing temperature
			A	4.8kb	60℃
B	4.3kb	60℃
			C	5.8kb	60℃
DPA	9.8kb	60℃
			DPB	9.1kb	60℃
DQA	7.3kb	60℃
			DQB	8.2kb	60℃
DRB	9.8kb	60℃

The enzyme used was Takara PrimerSTAR Max DNA polymerase, 50ng of template was taken and the extension time was 10 minutes.

2. Library construction

8 fragments of the same sample were quantified by Qubit (Invitrogen, USA) and mixed at an equimolar ratio, and then a library was constructed using DNA Template Prep Kit 4.0(Pacific Biosciences, USA).

3. Sequencing on machine

The constructed library was sequenced on the RSII (Pacbio) machine. The sequencing steps were performed according to the manufacturer's instructions.

4. Data analysis

Data analysis using the LAA module of SMRTlink 6.0 yielded high quality cluster (cluster) sequences, which were then used to determine genes. The results are shown in the following table.

HLA Gene statistics for each sample

	SHI001	SHI001-CM	DYR	DYR-CM
					A	2	2	2	3*
B	2	2	2	3*
					C	2	2	2	2
DPA	2	2	1	2*
					DPB	1	2*	1	5*
DQA	1	1	1	2*
					DQB	1	1	1	1
DRB	2	1*	1	3*

Note: is difference before and after directional differentiation

As can be seen, the HLA typing of iPS cells is different from that of cardiomyocytes differentiated therefrom. The HLA typing can be accurately carried out by detecting HLA of iPS cells and differentiated cells thereof.

Sequence listing

<110> technical research institute of marine industry

Shanghai Human Genome Research Center

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Claims

1. A primer whose amplification product comprises one or more fragments selected from the group consisting of: fragments of HLA-A gene amplified by using SEQ ID NOS: 1 and 2 as primers, fragments of HLA-B gene amplified by using SEQ ID NOS: 3 and 4 as primers, fragments of HLA-C gene amplified by using SEQ ID NOS: 5 and 6 as primers, fragments of HLA-DPA gene amplified by using SEQ ID NOS: 7 and 8 as primers, fragments of HLA-DPB gene amplified by using SEQ ID NOS: 9 and 10 as primers, fragments of HLA-DQA gene amplified by using SEQ ID NOS: 11 and 12 as primers, fragments of HLA-DQB gene amplified by using SEQ ID NOS: 13 and 14 as primers, and fragments of HLA-DRB gene amplified by using SEQ ID NOS: 15 and 16 as primers;

preferably, the primer is a sequence that hybridizes under stringent conditions to a fragment selected from one or more of the following: fragments of HLA-A gene amplified with SEQ ID NOS: 1 and 2 as primers, fragments of HLA-B gene amplified with SEQ ID NOS: 3 and 4 as primers, fragments of HLA-C gene amplified with SEQ ID NOS: 5 and 6 as primers, fragments of HLA-DPA gene amplified with SEQ ID NOS: 7 and 8 as primers, fragments of HLA-DPB gene amplified with SEQ ID NOS: 9 and 10 as primers, fragments of HLA-DQA gene amplified with SEQ ID NOS: 11 and 12 as primers, fragments of HLA-DQB gene amplified with SEQ ID NOS: 13 and 14 as primers, fragments of HLA-DRB gene amplified with SEQ ID NOS: 15 and 16 as primers,

more preferably, the primer comprises: (1) 1-16, and/or (2) a sequence having at least 90% identity to (1).

2. A probe for detecting an HLA gene, comprising:

fragments of HLA-A gene amplified by using SEQ ID NO 1 and 2 as primers,

fragments of HLA-B gene amplified by using SEQ ID NO. 3 and 4 as primers,

fragments of HLA-C gene amplified by using SEQ ID NO 5 and 6 as primers,

fragments of HLA-DPA gene amplified by using SEQ ID NOS 7 and 8 as primers,

fragments of HLA-DPB gene amplified by using SEQ ID NO 9 and 10 as primers,

the HLA-DQA gene fragment amplified by using SEQ ID NO 11 and 12 as primers,

the HLA-DQB gene fragment amplified by using SEQ ID NO 13 and 14 as primers,

(2) The complementary sequence of (1).

3. A kit comprising

(1) Primers for detecting HLA gene, and

optionally (2) a probe for detecting an HLA gene,

wherein the HLA gene is selected from one or more of the group consisting of: HLA-A, HLA-B, HLA-C, HLA-DPA, HLA-DPB, HLA-DQA, HLA-DQB, HLA-DRB,

preferably, the kit comprises the primers of claim 1, optionally the probes of claim 2, and optionally reagents required for PCR.

4. A method for HLA typing a differentiated cell of an iPS cell or reducing the risk of matching the differentiated cell of the iPS cell, comprising subjecting the differentiated cell of the iPS cell to one or more HLA gene tests and determining the genotype of the differentiated cell of the iPS cell based on the results of said HLA gene tests,

preferably, the HLA genes are selected from one or more of: HLA-A, HLA-B, HLA-C, HLA-DPA, HLA-DPB, HLA-DQA, HLA-DQB, and HLA-DRB.

5. The method of claim 4,

the HLA gene is detected by PCR amplification, and/or

The detection result of the HLA gene is the size and/or number of the amplified fragment.

6. The method of claim 5,

the HLA gene is detected as the number of amplified fragments comprising a nucleic acid sequence selected from one or more of: consisting of SEQ ID NO:1 and 2 as primers, and the sequence of about 4.8kb of the HLA-A gene consisting of SEQ ID NO:3 and 4 as primers, and the nucleic acid sequence of about 4.3kb of the HLA-B gene amplified by SEQ ID NO:5 and 6 as primers, the nucleotide sequence of about 5.8kb of HLA-C gene amplified by SEQ ID NO:7 and 8 as primers, the sequence of about 9.8k nucleic acid of HLA-DPA gene consisting of SEQ ID NO:9 and 10 as primers, the nucleic acid sequence of about 9.1kb of the HLA-DPB gene amplified from SEQ ID NO:11 and 12 as primers, the nucleic acid sequence of about 7.3kb of HLA-DQA gene amplified by SEQ ID NO:13 and 14 as primers, the nucleic acid sequence of about 8.2kb of HLA-DQB gene amplified by SEQ ID NO:15 and 16 as primers, and the amplified nucleic acid sequence of about 9.8kb of HLA-DRB gene.

7. Use of the primer according to claim 1 and the probe according to claim 2 for producing a kit for HLA typing a differentiated cell of an iPS cell or reducing the risk of matching a differentiated cell of an iPS cell,

preferably, the HLA typing comprises HLA gene testing of differentiated cells of iPS cells.

8. Use of the primer according to claim 1 and the probe according to claim 2 for producing a drug comprising differentiated cells of iPS cells,

preferably, the medicament is for the treatment of a disease benefiting from the differentiated cells of the iPS cells.

9. A method of amplifying HLA gene fragments of a sample, said HLA genes selected from one or more of: HLA-A, HLA-B, HLA-C, HLA-DPA, HLA-DPB, HLA-DQA, HLA-DQB, HLA-DRB, said amplification being performed using the primers of claim 1.

10. An apparatus for HLA-typing a differentiated cell of an iPS cell or reducing the risk of matching a differentiated cell of an iPS cell, the apparatus comprising a memory, a processor, and a computer program stored on the memory and executable on the processor, wherein the processor when executing the program implements the steps of:

(1) detecting one or more HLA genes of a differentiated cell of the iPS cell,

(2) determining the genotype of the differentiated cell of the iPS cell according to the result of the HLA gene detection,