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Link to original content: http://pubmed.ncbi.nlm.nih.gov/30711308/
Genetically-engineered pigs as sources for clinical red blood cell transfusion: What pathobiological barriers need to be overcome? - PubMed Skip to main page content
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Review
. 2019 May:35:7-17.
doi: 10.1016/j.blre.2019.01.003. Epub 2019 Jan 28.

Genetically-engineered pigs as sources for clinical red blood cell transfusion: What pathobiological barriers need to be overcome?

Benjamin Smood  1 , Hidetaka Hara  1 , Leah J Schoel  1 , David K C Cooper  2
Affiliations
Review

Genetically-engineered pigs as sources for clinical red blood cell transfusion: What pathobiological barriers need to be overcome?

Benjamin Smood et al. Blood Rev. 2019 May.

Abstract

An alternative to human red blood cells (RBCs) for clinical transfusion would be advantageous, particularly in situations of massive acute blood loss (where availability and compatibility are limited) or chronic hematologic diseases requiring frequent transfusions (resulting in alloimmunization). Ideally, any alternative must be neither immunogenic nor pathogenic, but readily available, inexpensive, and physiologically effective. Pig RBCs (pRBCs) provide a promising alternative due to their several similarities with human RBCs, and our increasing ability to genetically-modify pigs to reduce cellular immunogenicity. We briefly summarize the history of xenotransfusion, the progress that has been made in recent years, and the remaining barriers. These barriers include prevention of (i) human natural antibody binding to pRBCs, (ii) their phagocytosis by macrophages, and (iii) the T cell adaptive immune response (in the absence of exogenous immunosuppressive therapy). Although techniques of genetic engineering have advanced in recent years, novel methods to introduce human transgenes into pRBCs (which do not have nuclei) will need to be developed before clinical trials can be initiated.

Keywords: Blood transfusion; Pig, genetically-engineered; Red blood cells; Sickle cell disease; Xenotransfusion; Xenotransplantation.

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Conflict of interest statement

Conflicts of Interest Statement

The authors declare that they have no relevant conflicts of interest.

Figures

Figure 1:
Figure 1:. Human serum complement-dependent cytoxicity (CDC) of ABO-compatible human RBCs (ABO-C), ABO-incompatible human RBCs (ABO-I), wild-type pRBCs (WT), and GTKO pRBCs (GTKO)
Human sera (50%) of blood types O (n=10), A (n=9), B (n=8), and AB (n=4) were tested for CDC of human ABO-C, human ABO-I, pig WT, and pig GTKO RBCs. There was significantly greater lysis of WT than of ABO-I and GTKO RBCs (p<0.01). ABO-I RBCs sustained significantly greater lysis than of GTKO RBCs (p<0.01), but there was significantly greater lysis of GTKO than of ABO-C RBCs (**p<0.01). (Reproduced with permission from reference [52])
Figure 2:
Figure 2:. Phagocytosis of pRBCs is increased in GTKO-sensitized baboons
When GTKO-sensitized baboon serum (gray) was added to human and pig RBCs, there was significantly increased phagocytosis of WT and GTKO pRBCs, but decreased phagocytosis of human AB RBCs. When pooled human O serum (white) was added, human ABO-incompatible (AB) RBCs underwent greater phagocytosis than pRBCs. The small increase in phagocytosis of human group O RBCs likely reflects binding of baboon anti-human antibodies to the RBCs. *P<0.05, **P<0.01. (Modified with permission from reference [52])
Figure 3:
Figure 3:. Structures of human ABO and pig Gal glycans
Pig RBCs express Gal epitopes on oligosaccharides that are similar in structure to the human blood type B oligosaccharide, except for the fucose side-arm. (Reproduced with permission from reference [45])
Figure 4:
Figure 4:. Flow cytometric comparison of human IgM and IgG antibody binding to pig and human RBCs
Human IgM (left) and IgG (right) binding to WT, GTKO, GTKO/βGalNT2-KO (DKO), and GTKO/βGalNT2-KO/CMAHKO (TKO) pig RBCs and to human RBCs. The significant differences in human IgM and IgG binding to the various RBCs are indicated (*p<0.05, **p<0.01; ‡<0.05). There was no IgM/IgG binding to TKO pig or human RBCs (ns = not significant). (A relative Mean Fluorescence Intensity [MFI] <1 indicates no significant binding of IgM or IgG).
Figure 5:
Figure 5:. Flow cytometric comparison of human IgG and IgM antibody binding to pig and human RBCs
Human sera (n=83) were incubated with RBCs isolated from WT pigs (W), or from pigs lacking Gal (i.e., GTKO) and Neu5Gc (i.e., CMAH-KO) (double-knockout, D) or lacking Gal, Neu5Gc, and Sda (i.e., β4GalNT2-KO) (triple-knockout, T). These sera were also mixed with human (allogeneic) RBCs (H) expressing blood group O. Panels A and B show a summary of IgG and IgM binding to various RBCs, respectively. The data represent median fluorescent intensity (MFI). Panel C summarizes the number of samples from panels A and B where the indicated pRBC MFI is less than the MFI for the human blood group O RBCs. (Reproduced with permission from reference [67])
Figure 6:
Figure 6:. Protection from serum cytotoxicity provided by transgenic expression of human complement-regulatory proteins on nonactivated (left) and TNF-α-activated (right) pig corneal endothelial cells
Before activation, there was no serum cytotoxicity to GTKO, GTKO/CD46, or GTKO/CD46/CD55 pig cells. After activation, serum cytotoxicity was significantly increased, but the expression of two human complement-regulatory proteins (CD46 and CD55) almost completely prevented cytotoxicity.
Figure 7:
Figure 7:. Potential novel techniques for transgenic expression in pRBCs lacking nuclei
(A) CRISPR/Cas9 could be used to cleave genomic regions of GGTA1 (i.e., Gal) at sites flanking exons that contain the open reading frame (ORF). A replacement construct containing the ORF of CD47 flanked by GGTA1 sequences could be simultaneously introduced to facilitate the replacement of GGTA1 protein coding sequences with CD47. (B) The identical approach to that used in panel A could be repeated to replace codons encoding the CMAH gene with the CD55 ORF.
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