Growth hormone receptor knockout to reduce the size of donor pigs for preclinical xenotransplantation studies

Abstract

Background Many genetically multi‐modified donor lines for xenotransplantation have a background of domestic pigs with rapid body and organ growth. The intrinsic growth potential of porcine xeno‐organs may impair their long‐term function after orthotopic transplantation in non‐human primate models. Since growth hormone is a major stimulator of postnatal growth, we deleted its receptor (GHR‐KO) to reduce the size of donor pigs in one step. Methods Heart weight and proteome profile of myocardium were investigated in GHR‐KO and control pigs. GHR‐KO mutations were introduced using CRISPR/Cas9 in an α1,3‐galactosyltransferase (GGTA1)‐deficient background expressing the human cluster of differentiation (hCD46) and human thrombomodulin (hTHBD) to generate quadruple‐modified (4GM) pigs. Results At age 6 months, GHR‐KO pigs had a 61% reduced body weight and a 63% reduced heart weight compared with controls. The mean minimal diameter of cardiomyocytes was 28% reduced. A holistic proteome study of myocardium samples from the two groups did not reveal prominent differences. Two 4GM founder sows had low serum insulin‐like growth factor 1 (IGF1) levels (24 ± 1 ng/mL) and reached body weights of 70.3 and 73.4 kg at 9 months. Control pigs with IGF1 levels of 228 ± 24 ng/mL reached this weight range three months earlier. The 4GM sows showed normal sexual development and were mated with genetically multi‐modified boars. Offspring revealed the expected Mendelian transmission of the genetic modifications and consistent expression of the transgenes. Conclusion GHR‐KO donor pigs can be used at an age beyond the steepest phase of their growth curve, potentially reducing the problem of xeno‐organ overgrowth in preclinical studies.

Cardiac Xenotransplantation: Progress in Preclinical Models and Prospects for Clinical Translation

“…Recently, Hinrichs et al have produced GHRKO pigs in order to address intrinsic organ growth. They demonstrate that GHRKO pigs have slow or reduced growth, including their organs’ growth, compared to normal wild-type pigs ( 61 , 104 – 107 ). Recently, Goerlich, et al have examined intrinsic and extrinsic causes of graft growth after transplantation in an OHTx model using “multi-gene” pigs growth hormone receptor knockout pigs (GHRKO) ( 69 ).…”

An intrinsic link to an extrinsic cause of cardiac xenograft growth after xenotransplantation

“…Genetically modified swine with antigenic knockouts and human transgene knock‐ins are used as xenograft donors, along with CD40/CD40L co‐stimulation blockade, in order to produce xenografts that are immunocompatible for human transplantation 7 . GHRKO in pigs as an additional knockout has been proposed to address intrinsic factors of graft growth; namely, the exponential growth seen in age‐matched xenograft donors 8 . Similar to mouse models, GHRKO pigs are characterized by decreased circulating IGF‐1, proportional growth retardation, and mirror the human disease Laron syndrome, with a similar phenotype 9 …”

“…7 GHRKO in pigs as an additional knockout has been proposed to address intrinsic factors of graft growth; namely, the exponential growth seen in age-matched xenograft donors. 8 Similar to mouse models, GHRKO pigs are characterized by decreased circulating IGF-1, proportional growth retardation, and mirror the human disease Laron syndrome, with a similar phenotype. 9 We demonstrated that the addition of GHRKO to these genetically engineered cardiac xenografts, in order to reduce xenograft growth, prolongs survival up to 9 months (264 days).…”

Current status of the application of gene editing in pigs

“…Xeno-organs donated by genetically modified pigs carrying the genetic background of domestic pigs can grow rapidly; this can generate incompatibility with recipients and impair their long-term function after transplantation. Growth hormone receptor ( GHR )-deficient pigs with reduced organ size [ 73 , 74 ] and subsequent GHR / GGTA1 double-knockout pigs expressing the human cluster of differentiation ( hCD46 ) and human thrombomodulin ( hTHBD ) [ 75 ] have been generated by gene editing. This approach will improve the implementation of xenotransplantation.…”