Researchers modify more than 60 pig genes in effort to enable organ transplants into human

For decades, scientists and doctors have dreamed of creating a steady supply of human organs for transplantation by growing them in pigs. But concerns about rejection by the human immune system and infection by viruses embedded in the pig genome have stymied research. Now, by modifying more than 60 genes in pig embryos — ten times more than have been edited in any other animal — researchers believe they may have produced a suitable non-human organ donor.

Geneticist George Church of Harvard Medical School in Boston, Massachusetts, announced that he and colleagues had used the CRISPR/Cas9 gene-editing technology to inactivate 62 porcine endogenous retroviruses (PERVs) in pig embryos. These viruses are embedded in all pigs’ genomes and cannot be treated or neutralized. It is feared that they could cause disease in human transplant recipients.

Church’s group also modified more than 20 genes in a separate set of pig embryos, including genes that encode proteins that sit on the surface of pig cells and are known to trigger a human immune response or cause blood clotting. Church declined to reveal the exact genes, however, because the work is as yet unpublished. Eventually, pigs intended for organ transplants would need both these modifications and the PERV deletions.

Preparing for implantation

“This is something I’ve been wanting to do for almost a decade,” Church says. A biotech company that he co-founded to produce pigs for organ transplantation, eGenesis in Boston, is now trying to make the process as cheap as possible.

Church released few details about how his team managed to remove so many pig genes. But he says that both sets of edited pig embryos are almost ready to implant into mother pigs. eGenesis has procured a facility at Harvard Medical School where the pigs will be implanted and raised in isolation from pathogens.

eGenesis is a life sciences company whose mission is to transform xenotransplantation into an everyday, lifesaving ​medical procedure.

Science – Genome-wide inactivation of porcine endogenous retroviruses (PERVs)

The shortage of organs for transplantation is a major barrier to the treatment of organ failure. While porcine organs are considered promising, their use has been checked by concerns about transmission of porcine endogenous retroviruses (PERVs) to humans. Here, we describe the eradication of all PERVs in a porcine kidney epithelial cell line (PK15). We first determined the PK15 PERV copy number to be 62. Using CRISPR-Cas9, we disrupted all 62 copies of the PERV pol gene and demonstrated a over a 1000-fold reduction in PERV transmission to human cells using our engineered cells. Our study shows that CRISPR-Cas9 multiplexability can be as high as 62 and demonstrates the possibility that PERVs can be inactivated for clinical application to porcine-to-human xenotransplantation.

46 pages of supplemental information for Genome-wide inactivation of porcine endogenous retroviruses (PERVs)

List of eGenesis papers

Genome-wide inactivation of porcine endogenous retroviruses (PERVs)

Yang L, Güell M, Niu D, George H, Lesha E, Grishin D, Aach J, Shrock E, Xu W, Poci J, Cortazio R, Wilkinson RA, Fishman JA, Church GM (2015). Genome-wide inactivation of porcine endogenous retroviruses (PERVs) Science (in press) – advance of print

Chari R, Mali P, Moosburner M, Church GM (2015). Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nature Methods (in press)

Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL, Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM (2015). Highly efficient Cas9-mediated transcriptional programming. Nature Methods. 12(4):326-8. PMID: 25730490

Yang L, Grishin D, Wang G, Aach J, Zhang CZ, Chari R, Homsy J, Cai X, Zhao Y, Fan JB, Seidman C, Seidman J, Pu W, Church G (2014). Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells. Nature Communications. PMID: 25425480

Byrne SM, Ortiz L, Mali P, Aach J, Church GM (2014). Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Nucleic Acids Research. 43(3):e21. PMID: 25414332

Byrne SM, Mali P, Church GM (2014). Genome editing in human stem cells. Methods in Enzymology. 546:119-38. PMID:25398338

Esvelt KM, Smidler AL, Catteruccia F, Church GM (2014). Concerning RNA-guided gene drives for the alteration of wild populations. eLife. PMID: 25035423

Yang L, Yang JL, Byrne S, Pan J, Church GM (2014). CRISPR/Cas9-Directed Genome Editing of Cultured Cells. Current Protocols in Molecular Biology. 107:31.1.1-31.1.17. PMID: 24984853

Aach J, Mali P, Church GM (2014). CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes. BioRxiv. Link

Yaung SJ, Esvelt KM, Church GM (2014). CRISPR/Cas9-mediated phage resistance is not impeded by the DNA modifications of phage T4. PLoS One. PMID: 24886988

​Wang G, McCain ML, Yang L, He A, Pasqualini FS, Agarwal A, Yuan H, Jiang D, Zhang D, Zangi L, Geva J, Roberts AE, Ma Q, Ding J, Chen J, Wang DZ, Li K, Wang J, Wanders RJ, Kulik W, Vaz FM, Laflamme MA, Murry CE, Chien KR, Kelley RI, Church GM, Parker KK, Pu WT (2014) Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies.Nature Medicine. 20(6):616-23. PMID: 24813252

Guell M, Yang L, Church G (2014) Genome Editing Assessment using CRISPR Genome Analyzer (CRISPR-GA) Bioinformatics. PMID: 24990609

Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E (2014). CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Research. PMID: 24861617

Yang L, Mali P, Kim-Kiselak C, Church G (2014). CRISPR-Cas-mediated targeted genome editing in human cells. Methods in Molecular Biology. 1114:245-67. PMID: 24557908

Mali P, Esvelt KM, Church GM (2013). Cas9 as a versatile tool for engineering biology. Nature Methods. 10(10):957-63. PMID:24076990

Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM (2013). Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature Methods. 10(11):1116-21. PMID: 24076762

Tzur YB, Friedland AE, Nadarajan S, Church GM, Calarco JA, Colaiácovo MP (2013). Heritable custom genomic modifications in Caenorhabditis elegans via a CRISPR-Cas9 system. Genetics. 195(3):1181-5. PMID: 23979579

Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J (2013). Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature Biotechnology. 31(8):688-91. PMID: 23929339

Yang L, Guell M, Byrne S, Yang JL, De Los Angeles A, Mali P, Aach J, Kim-Kiselak C, Briggs AW, Rios X, Huang PY, Daley G, Church G (2013). Optimization of scarless human stem cell genome editing. Nucleic Acids Research. 41(19):9049-61. PMID:23907390

Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM (2013). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 31(9):833-8. PMID: 23907171

Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA (2013). Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nature Methods. 10(8):741-3. PMID: 23817069

DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013). Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Research. 41(7):4336-43. PMID: 23460208

Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013). RNA-guided human genome engineering via Cas9. Science. 339(6121):823-6. PMID: 23287722

SOURCE – Journal Science, Nature, eGenesis

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