Sanger HLA Typing

Sanger HLA Typing

1. Background Introduction

1.1 Project Background

With the rapid development of allogeneic immune cell therapy, the universal (off-the-shelf) CAR-T therapy has become an important breakthrough direction in tumor immunotherapy. Compared with autologous CAR-T, universal CAR-T has significant advantages such as "ready supply" and large-scale production, capable of significantly reducing costs, shortening cycles, and providing timely therapeutic opportunities for more patients. However, one of the core challenges facing this revolutionary technology is how to overcome graft-versus-host disease (GvHD) from immune rejection, with the Human Leukocyte Antigen (HLA) system being the key factor in this challenge.

During the development of universal CAR-T, gene editing technologies such as CRISPR/Cas9 are typically needed to remove T cell TCR and HLA-I molecules, and even to knock out immune checkpoint molecules anti-host (GvHD) genes. To ensure accurate editing, avoid off-target effects and potential editing risks, it is necessary to first obtain precise HLA typing information at the target locus. Subsequently, guide RNA (gRNA) design with continuous safety verification provides an indispensable foundation.

Figure 1: Core Application of HLA Precision Typing in Universal CAR-T Cell Development

1.2 Two Key Reasons for Absolute Necessity of Four-Digit Typing

Traditional HLA typing mainly meets the requirements of transplant migration, and can be achieved at two-digit typing. However, gene editing in universal CAR-T imposes higher requirements on HLA typing precision. Four-digit high-resolution typing (such as HLA-A*02:01:01:01) can accurately identify synonymous mutations in coding regions and non-synonymous mutations in non-coding regions. This seemingly minor difference may have a critical impact on sgRNA design and gene editing efficiency, and must be carefully considered in the following two aspects:

(1) Reason 1 - sgRNA Design Precision Requirements:

The editing specificity of CRISPR/Cas9 gene editing depends highly on the precise matching between sgRNA and the target sequence. Even the same "family" (such as A*02) HLA genes can have multiple variants in single nucleotide polymorphism (SNP) positions in their sequences.

(2) Reason 2 - Regulatory Audit Strict Standards:

With the maturity of gene editing technology, global drug regulatory agencies have increasingly strict requirements for universal CAR-T cell therapy product applications and approval, increasingly emphasizing the accuracy of multi-layered high-level precision.

Therefore, providing four-digit high-resolution HLA typing data based on gold-standard methods is gradually becoming a "hard requirement" in the IND application reports for universal CAR-T products.

Figure 2: Dual Requirements for Four-Digit High-Resolution HLA Typing in Universal CAR-T Product Development

2. Technical Principles and Workflow

Although NGS and TGS technologies are continuously developing, Sanger sequencing is still recognized for its proximity to the gold-standard accuracy, and is regarded by the global academic community and regulatory agencies as the "gold standard" for gene sequence validation. In application scenarios where HLA four-digit high-resolution typing accuracy needs to meet demanding requirements, Sanger sequencing remains the most reliable option. The most authoritative choice.

2.1 Homozygous Sample Detection Workflow

For homozygous samples, in which the two HLA alleles at the same locus are completely identical, we use direct sequencing to compare the strategies, which is simple, fast, and efficient.

(1) gDNA extraction and quality control: Extract high-quality genomic DNA from the sample;

(2) PCR amplification: Use primers specific to HLA genes to perform PCR amplification on core regions;

(3) Product purification and sequencing: Perform full-length sequencing on purified PCR products using Sanger sequencing method;

(4) Data analysis: Compare the sequencing results with IPD-IMGT/HLA database for comparison, obtaining high-resolution typing results for four digits.

Figure 3: Homozygous HLA Typing Workflow

2.2 Heterozygous Sample Typing Workflow

For heterozygous samples, in which the two alleles at the same locus contain different HLA allele genes, direct sequencing generates overlapping signal peaks that cannot be accurately resolved. Therefore, we adopt a classic allele cloning technique, physically isolating two different allele genes for typing and separate sequencing, thereby ensuring the uniqueness of the single-peak results.

(1) gDNA extraction and PCR amplification: Same as homozygous workflow;

(2) TA cloning: After PCR product is purified to the T vector, construct gene tissue library;

(3) Transformation and screening: Transform tissue to attentive state during intense enrichment, select white colonies through blue-white screening;

(4) Monoclonal culture and sequencing: Culture the collected monoclonal performing Sanger sequencing, as each clone only contains one equal-position gene copy, it can obtain clean and single-peak results;

(5) Data analysis: Compare the sequencing results for analysis of two different equal-position genes, ultimately determining the complete four-digit high-resolution typing for heterozygotes.

Figure 4: Heterozygous HLA Typing Workflow

3. Technical Advantages

4. Application Scenarios

(1) CAR-T and gene editing cell point sequence confirmation: Provide accurate HLA locus sequence target for gene editing projects aimed at specific target sequences to establish accurate gold-standard basis;

(2) Difficulty in typing result verification: Used as a verification method to supplement typing confirmation for NGS or TGS high-throughput typing emergent situations where new alleles or ambiguous results exist;

(3) Classic migration configuration: Provide high-resolution typing for HLA-A, B, C, DRB1, DQB1 loci to meet the requirements of clinical bed accuracy;

(4) Drug gene group research: Accurately determine the specific HLA and drug response-related equal-position genes.

5. Demonstration Report

We provide professional and standardized HLA typing reports that meet clinical diagnostics and research requirements. Reports present typing results in tabular detailed sequence alignment comparison format, and align with the latest database and nomenclature of the International Immunogenetics Project (IPD-IMGT/HLA) [2].

Figure 5: Sanger HLA Typing Report Examples, Including Typing Results and Sequencing Peaks

6. Service Content and Sample Requirements 6.1 Service Content

*Service Cycle: 10 working days for homozygous standard workflow; 20 working days for heterozygous workflow.

6.2 Sample Requirements

To ensure the accuracy and reliability of detection results, please strictly follow the standards below to prepare and send samples.

6.3 Sample Information and Transport

(1) Information requirements: Each sample should be clearly labeled with sample number, and upload the sending list, note sample type, source and other relevant information;

(2) Transport guidelines: Please use foot dry ice (for frozen samples) or blue ice (for cold samples) for transport, and select reliable fast services to ensure samples are transported during the stable period.

Note: If the sample does not meet the requirements, it may affect data quality, extend detection cycle or lead to failure. If you have special sample types, please contact our technical support team in advance.

7. References

[1] Mack M, Miaskowski C, Bhat A, et al. Performance characteristics and validation of next-generation sequencing for human leucocyte antigen typing. J Mol Diagn. 2016;18(5):668-675.

[2] Robinson, J., et al. (2020). The IPD-IMGT/HLA Database. Nucleic Acids Research, 48(D1), D948-D955.

[3] Liu P, Yao M, Gong Y, et al. Benchmarking the human leukocyte antigen typing performance of three assays and seven next-generation sequencing-based algorithms. Front Immunol. 2021;12:652258.