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Research Highlights

Muller, Elmi MBChB, MMED1

doi: 10.1097/TP.0000000000001918
In View: Research Highlights

1 Groote Schuur Hospital, Main Road, Observatory, Cape Town, Western Cape, South Africa.

Received 14 August 2017.

Accepted 15 August 2017.

The author declares no funding conflicts of interest.

Correspondence: Dr. Elmi Muller, MBChB, MMED, Groote Schuur Hospital, Main Road, Observatory, Cape Town, Western Cape, 7925, South Africa. (

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Inactivation of Porcine Endogenous Retrovirus in Pigs Using CRISPR-Cas9

Niu D, Wei HJ, Lin L, et al. [published online August 10 2017]. Science. doi: 10.1126/science.aan4187

Targeted changes to the genome of a living cell had become a reality with the discovery of the clustered regularly interspaced short palindromic repeats (CRISPR) system originally discovered in bacteria and archaea, enabling these organisms to eliminate invading genetic material. The system allows foreign DNA to be incorporated into the bacterial genome at the CRISPR loci which is then transcribed and processed into RNA targeting site-specific DNA. Recently, genome editing has been widely adopted to target important genes.1

In this study, the authors demonstrated the feasibility of applying this technique to eliminate the cross-species transmission of porcine endogenous retroviruses (PERVs). The authors firstly demonstrated that PERVs from pig epithelial cell lines do infect human embryonic kidney cells if cocultured for a week. They then observed the cells for a period of 4 months and found that the copy number PERVs increased over time secondary to intracellular transposition, or, alternatively, by intercellular PERV transmission. Thus, infected human cells can transmit PERVs to previously unexposed human cells.

Next, the authors inactivated PERV activity in a primary porcine fetal fibroblast line (FFF3) by designing 2 CRISPR guided RNAs specific to the catalytic core of the PERV pol gene. The authors failed to obtain a 100% PERV-inactivated FFF3 clone initially. However, when expanding the FF3 clones, they achieved a 100% PERV inactivation by treating them with a combination of a p52 inhibitor PFTα and bFGF. Highly modified clones survived under this condition.

Lastly, the authors examined a genotyped PERV-inactivated pig by deep sequencing of the PERV pol loci and demonstrated a 100% PERV eradication. These PERV-inactivated animals have the potential to address major safety concerns in clinical xenotransplantation in the future.

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1. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819–23.

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HLA-E-expressing Pluripotent Stem Cells Escape Allogeneic Responses and Lysis by NK Cells

Gornalusse GG, Hirata RK, Funk SE, et al. Nat Biotechnol. 2017;35(8):765–72.

Human pluripotent stem cells (PSCs) have the potential to treat many human diseases, but their clinical use is limited with the expression of highly polymorphic HLA genes. Here, the authors explore the potential of a single PSC line that can avoid allogeneic rejection.

The beta-2 microglobulin (B2M) gene encodes the HLA class 1 surface expression initiating the allogeneic rejection process with the stimulation of CD8+ T cells. The authors created B2M-negative PSCs (B2M−/−), but found that the HLA class I–negative cells are still lysed by natural killer (NK) cells.

NK cell–dependent lysis is normally inhibited by the HLA-E protein. Most human NK cells express NKG2A, an inhibitory receptor that binds to HLA-E. By engineering HLA-E allogeneic CD8+ T cells, the authors created a cell line that is not only resistant to NK-mediated lysis but also unable to be recognized as allogeneic. This was done by developing a B2M−/− cell line that expresses HLA-E as a single chain protein using a recombinant adenoassociated virus–mediated gene editing vector.

This work successfully demonstrates that single-chain HLA-E molecules prevent the NK-mediated lysis of B2M−/− cells without stimulating allogeneic T cells, addressing a major problem in the creation of universal donor cells for regenerative medicine applications. The next step would be to look at the impact of reduced HLA expression on clearing infections and developing malignancies.

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