Introduction: Xenotransplantation has been considerably advanced by identifying the genetic modifications needed for the donor pig. These include (1) triple knockout (KO) (TKO) of xenoantigen encoding genes:- GGTA1, B4GaLNT2 and CMAH, (2) KO of GHR, to limit organ growth, and (3) addition of human genes: (i) complement inhibitors (CIs), CD46/CD55/CD59; (ii) thromboregulatorsy hTBM/hEPCR; (iii) apoptosis inhibitors hHO-1(iv) a macrophage inhibitor hCD47. However, challenges remain in generating multi-transgenic pigs for production in a time- and cost-efficient manner. The aim of this study is to generate multi-transgenic donor pigs with ten genetic edits (10GE), introducing six essential human transgenes as a single multi-cistronic  vector (MCV) in one step.
Methods: We constructed five MCVs, each containing six human transgenes organized into three bicistronic units, utilizing the 2A self-cleaving peptide system.   Each transgene pair was linked by a 2A peptide, to  allowing the expression of both transgenes from a single promoter. Homology arms were incorporated to facilitate Homology Directed Repair (HDR) at the CMAH or GGTA1/Neo locus landing pads.
Each MCV was transfected as a linear fragment (~28kb), flanked by HDR arms, into GGTA1 knockout fibroblasts, along with the appropriate CRISPR guides and Cas9. Vector targeting was confirmed by PCR, ddPCR, and Southern blot. Transgene expression was evaluated in heart, lung, and kidney tissue samples using an automated capillary western blot assay system and immunohistochemistry (IHC). Transgene function in aortic endothelial cells was confirmed in vitro by Complement–Dependent Cytotoxicity (CDC) assay (hCD46, hDAF), Activated Protein C (APC) assay (hTBM, hEPCR) and SIRP-areceptor activation assay (hCD47)
Results: No significant differences in transgene expression were observed between the two landing pads in the 10GE pigs. Physiological levels of hTBM and hEPCR were low when co-expressed in a bi-cistron controlled by pTBMp, while expression of hCD55, hCD46, and hCD47 was robust at physiologically acceptable levels under CAGp. hHO-1 levels remained low regardless of the upstream gene. hCD47 expression was strongest when placed immediately downstream of the CAG promoter, but was barely detectable when positioned second, downstream of the 2A sequence. Nevertheless, In vitro assays showed that hCD47 protein expressed in endothelial cells  from different MCVs retained the ability to bind and activate SIRP-α, albeit to varying degrees. MCVs expressing two (hCD46 & hCD55) or three (hCD46, hCD55 and hCD59) CIs performed equally in CDC assays. In all 10GE genotypes had similarAPC levels were comparable to a human endothelial cell line. 
Conclusion: To date, live pigs have been generated with four of these MCVs, and organs from three 10GE genotypes have been used in xenotransplantation experiments toin baboons and human recipients.