Development of a BM7G((TKO/hCD46/hCD55/hTHBD/hEPCR) Donor Pig with Endogenous Promoter-Driven Transgenes for Xenotransplantation

This study successfully developed a seven-gene modified donor pig (BM7G) by combining CRISPR-Cas9-mediated knockout of three glycan antigens with site-specific integration of four human protective genes driven by endogenous promoters, resulting in a model with stable, tissue-specific expression that effectively reduces immunogenicity and thrombosis for xenotransplantation.

The Gist

Imagine a world where the shortage of human organs for transplants is solved by using organs from pigs. It sounds like science fiction, but scientists are making it a reality. However, there's a big problem: if you put a pig organ into a human body, the human immune system attacks it immediately, like a fortress firing cannons at an intruder. This is called "rejection."

This paper describes how a team of scientists in China built a special "super-pig" (named BM7G) designed to be a safe, long-term organ donor for humans. They did this by giving the pig a genetic "makeover" with seven specific changes.

Here is the story of how they did it, explained with simple analogies:

1. The Problem: The "Red Flag" and the "Missing Shield"

Think of a pig cell as a house.

• The Red Flags (Xenoantigens): The pig's natural cells have three specific "flags" on their surface (called $\alpha$Gal, Neu5Gc, and Sda). To the human immune system, these flags scream "INTRUDER!" and trigger an immediate, violent attack (Hyperacute Rejection).
• The Missing Shield: Even if you remove the flags, the pig's cells still lack the "security guards" that human cells have to stop the immune system from attacking.

2. The Solution: A Seven-Step Genetic Makeover

The scientists performed two main types of surgery on the pig's DNA:

Step A: Erasing the Red Flags (The Knockout)

First, they used a molecular pair of scissors called CRISPR-Cas9 to cut out the three genes responsible for making those "Red Flags."

• The Result: The pig cells no longer display the signals that trigger the human immune system's immediate panic. It's like painting over the "Intruder" sign so the immune system doesn't notice the house at first.

Step B: Installing Human Security Guards (The Knock-in)

Next, they needed to add human "security guards" (proteins) to the pig cells to stop the immune system from attacking later. They wanted to insert four human genes:

1. hCD55 & hCD46: These stop the immune system from blowing up the cells (Complement regulation).
2. hTHBD & hEPCR: These stop the blood from clotting inside the new organ (Coagulation regulation).

The Innovation: The "Safe Harbor" and the "Local Manager"
Usually, when scientists insert new genes, they use a generic "on-switch" (an exogenous promoter) to make the gene work. But this is risky; the pig's body might eventually realize this switch is foreign and turn it off (like a landlord evicting a tenant).

Instead, this team used a clever trick:

• The Safe Harbor (Rosa26 Locus): They inserted the new genes into a specific, safe spot in the pig's DNA called the Rosa26 locus. Think of this as a VIP parking spot in a garage where cars are never towed.
• The Local Manager (Endogenous Promoters): Instead of using a foreign "on-switch," they used the pig's own natural switches to turn on the new human genes.
  • For the general security guards (CD55/CD46), they used the Rosa26 switch, which is active everywhere in the body (like a master key).
  • For the blood-clotting guards (THBD/EPCR), they used the THBD switch, which only turns on in blood vessels (like a specialized key for the front door).

Why is this better? Because the pig's body recognizes these switches as "native," it won't try to silence them. This ensures the human proteins stay active for the pig's entire life, not just for a few weeks.

3. Cleaning Up the Construction Site

When they first built this pig, they had to leave a "construction marker" (a selection gene) to help them find the successful edits. But you don't want a construction marker left on a finished house.

• They used a tool called Cre/loxP to act like a cleanup crew, snipping out the temporary marker and leaving only the clean, modified DNA behind.

4. The Results: A Successful Test Drive

The scientists tested these "BM7G" pigs in the lab:

• No Red Flags: They confirmed the three "Intruder" signals were completely gone.
• Active Guards: They found that the human security proteins were working perfectly in the pig's heart, liver, and kidneys.
• Human Blood Test: When they mixed the pig's cells with human blood:
  • Human antibodies didn't stick to the pig cells.
  • The pig cells didn't get destroyed by the human immune system.
  • The pig cells successfully prevented human blood from clotting on them.

The Big Picture

This paper isn't just about making a pig; it's about solving a stability problem. Previous attempts often failed because the added human genes would eventually turn off. By using the pig's own "on-switches" (endogenous promoters), these scientists created a pig that is genetically stable and ready to be a reliable organ donor.

In short: They took a pig, removed the parts that make humans reject it, and installed human "bodyguards" using the pig's own instruction manual to ensure those guards never clock out. This brings us one step closer to a future where we can save lives with pig organs.

Technical Summary

1. Problem Statement

Xenotransplantation using genetically modified pigs is a promising solution to the global organ shortage. However, current strategies face two critical limitations regarding long-term stability and efficacy:

• Unstable Expression: Transposon-based methods (e.g., PiggyBac) often result in random integration, leading to copy number variations and unstable inheritance.
• Epigenetic Silencing: Even with site-specific integration (knock-in), the use of exogenous promoters (e.g., viral promoters like CMV or CAG) often triggers host epigenetic silencing mechanisms. This leads to the gradual attenuation of transgene expression over time, compromising the long-term survival of xenografts.

There is a critical need for a donor pig model that combines the elimination of major xenoantigens with the stable, long-term, and tissue-specific expression of human protective genes without the risk of silencing.

2. Methodology

The researchers developed a seven-gene-modified (7G) Bama miniature pig (termed BM7G) using a multi-step gene-editing workflow:

• Triple Knockout (TKO):
  • Target: GGTA1, CMAH, and β4GALNT2 genes (responsible for synthesizing $\alpha$-Gal, Neu5Gc, and Sda antigens).
  • Method: CRISPR-Cas9 mediated knockout in porcine fetal fibroblasts (PFFs) to eliminate hyperacute rejection.
• Site-Specific Knock-In (Rosa26 Locus):
  • Target: The porcine Rosa26 safe-harbor locus.
  • Strategy: Homology-Directed Repair (HDR) was used to insert a dual-expression cassette containing four human protective genes:
    1. hCD55 and hCD46 (Complement regulators).
    2. hTHBD (Thrombomodulin) and hEPCR (Endothelial Protein C Receptor) (Coagulation regulators).
  • Promoter Strategy (Key Innovation):
    • hCD55 and hCD46 were driven by the endogenous porcine Rosa26 promoter to ensure ubiquitous, stable expression.
    • hTHBD and hEPCR were driven by the endogenous porcine THBD core promoter to ensure vascular endothelial-specific expression.
  • Selection Marker Removal: A PGK-Puro selection cassette flanked by loxP sites was inserted. After selection, Cre/loxP recombination was used to excise the marker, leaving a "clean" genome for clinical safety.
• Animal Production:
  • Gene-edited PFFs were used for Somatic Cell Nuclear Transfer (SCNT) to generate cloned piglets.
  • Multiple rounds of cloning were performed: first to generate TKO pigs, then to insert the Rosa26 cassette, and finally to remove the selection marker.

3. Key Contributions

• Endogenous Promoter Strategy: The study successfully demonstrated that using endogenous promoters (Rosa26 and THBD) to drive human transgenes prevents epigenetic silencing, a common failure point in previous xenotransplantation models.
• Synergistic Multi-Gene Modification: The creation of a "7G" pig (3 knockouts + 4 knock-ins) that simultaneously addresses hyperacute rejection, complement activation, and coagulation dysregulation.
• Marker-Free Model: The successful removal of the antibiotic selection marker via Cre/loxP, reducing immunogenicity risks and regulatory hurdles for clinical translation.
• Tissue-Specific Expression Control: The model achieved a precise expression profile: ubiquitous expression of complement regulators and endothelial-specific expression of coagulation regulators, mimicking the natural physiological distribution of these proteins.

4. Key Results

• Genetic Verification:
  • Genotyping confirmed the complete biallelic knockout of GGTA1, CMAH, and β4GALNT2.
  • PCR and sequencing confirmed precise integration at the Rosa26 locus and successful excision of the selection marker.
  • Off-Target Analysis: Sanger sequencing of predicted off-target sites for all gRNAs revealed zero off-target mutations.
• Protein Expression:
  • Flow Cytometry: BM7G porcine cells showed a complete absence of $\alpha$-Gal, Sda, and Neu5Gc antigens.
  • Expression Patterns:
    • hCD55 and hCD46 were widely expressed in vascular endothelial cells (PECs) and fibroblasts (PEFs), comparable to human levels.
    • hTHBD and hEPCR showed strict vascular specificity, expressed in PECs but absent in fibroblasts, confirming the efficacy of the THBD promoter.
  • Organ Level: Western blot, RT-qPCR, and immunofluorescence confirmed stable expression of all four human proteins in heart, liver, and kidney tissues.
• Functional Assays (In Vitro):
  • Antibody Binding: BM7G cells showed significantly reduced binding of human IgM and IgG compared to wild-type (WT) and TKO cells.
  • Complement-Dependent Cytotoxicity (CDC): BM7G endothelial cells exhibited near-zero cell death when exposed to human serum, demonstrating effective complement regulation by hCD55/hCD46.
  • Coagulation (TAT Assay): BM7G endothelial cells significantly reduced Thrombin-Antithrombin (TAT) complex formation when co-cultured with human blood, indicating effective regulation of the coagulation cascade by hTHBD/hEPCR.

5. Significance

This paper presents a robust, preclinical-grade donor pig model that addresses the critical bottleneck of transgene stability in xenotransplantation. By replacing exogenous viral promoters with endogenous porcine promoters, the authors have created a model capable of sustained, long-term expression of protective human genes.

The BM7G pig offers a synergistic defense mechanism:

1. Elimination of major carbohydrate antigens to prevent hyperacute rejection.
2. Stable expression of complement inhibitors to prevent acute vascular rejection.
3. Endothelial-specific expression of coagulation regulators to prevent thrombotic microangiopathy.

This model provides a superior resource for preclinical pig-to-non-human primate studies and brings the field closer to the clinical translation of xenotransplantation, potentially solving the organ shortage crisis with a safer, more reliable donor source.