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Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation.

ABSTRACT: Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies.

SUBMITTER: Cabanes-Creus M

PROVIDER: S-EPMC10940703 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation.

Cabanes-Creus Marti M Liao Sophia H Y SHY Gale Navarro Renina R Knight Maddison M Nazareth Deborah D Lau Ngee-Soon NS Ly Mark M Zhu Erhua E Roca-Pinilla Ramon R Bugallo Delgado Ricardo R Vicente Ana F AF Baltazar Grober G Westhaus Adrian A Merjane Jessica J Crawford Michael M McCaughan Geoffrey W GW Unzu Carmen C González-Aseguinolaza Gloria G Alexander Ian E IE Pulitano Carlo C Lisowski Leszek L

Nature communications 20240314 1

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in hum ...[more]

PMID: 38485924

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Project description:BackgroundHuman albumin/dextran (HA-D), bovine-gelatin (BG), and packed red blood cells plus plasma have been used in European and North-American clinical trials of normothermic ex situ liver perfusion (NEsLP). We compared the effects of these perfusates in a porcine model during NEsLP and after transplantation.MethodsPorcine livers were retrieved 30 minutes after circulatory death. After 5 hours of NEsLP, grafts were transplanted. Three groups (n = 6) were assessed (HA-D vs BG vs whole blood [WB]). One group of static cold storage (SCS) was evaluated for comparison with the perfusion groups. Hemodynamic variables, liver and endothelial injury, and function were assessed during NEsLP and posttransplantation.ResultsHepatic artery flow was higher since the beginning of NEsLP in the HA-D group (HA-D, 238 ± 90 mL/min vs BG, 97 ± 33 mL/min vs WB, 148 ± 49 mL/min; P = 0.01). Hyaluronic acid was lower in the HA-D at the end of perfusion (HA-D, 16.28 ± 7.59 ng/μL vs BG, 76.05 ± 15.30 ng/μL vs WB, 114 ± 46 ng/μL; P < 0.001). After transplant, aspartate aminotransferase was decreased in the HA-D group when compared with the rest of the groups (HA-D, 444 ± 226 IU/L vs BG, 1033 ± 694 IU/L vs WB, 616 ± 444 IU/L vs SCS, 2235 ± 1878 IU/L). At 5 hours after transplant, lactate was lower in the HA-D group (HA-D, 3.88 ± 1.49 mmol/L vs BG, 7.79 ± 2.68 mmol/L vs WB, 8.16 ± 3.86 mmol/L vs SCS, 9.06 ± 3.54 mmol/L; P = 0.04). International Normalized Ratio was improved in HA-D group compared to the rest of the groups (HA-D, 1.23 ± 0.30 vs BG, 1.63 ± 0.20 vs WB, 1.50 ± 0.31 vs SCS, 1.97 ± 1.55; P = 0.03) after transplantation. In contrast, BG displayed lower aspartate aminotransferase levels during NEsLP (HA-D, 183 ± 53 IU/L vs BG, 142 ± 52 IU/L vs WB, 285 ± 74 IU/L; P = 0.01) and less cleaved-caspase-3 staining (HA-D, 2.05 ± 0.73% vs BG, 0.95 ± 1.14% vs WB, 1.74 ± 0.54% vs SCS, 7.95 ± 2.38%) compared with the other groups. On the other hand, the bile from the WB showed higher pH (HA-D, 7.54 ± 0.11 vs BG, 7.34 ± 0.37 vs WB, 7.59 ± 0.18) and lower glucose levels (HA-D, 0.38 ± 0.75 mmol/L vs BG, 1.42 ± 1.75 mmol/L vs WB, 0 ± 0 mmol/L) by the end of perfusion.ConclusionsOverall HA-D displayed more physiologic conditions during NEsLP that were reflected in less graft injury and improved liver function and survival after transplantation. Optimization of the perfusates based on the beneficial effects found with these different solutions would potentially improve further the outcomes through the use of NEsLP in marginal grafts.

Project description:IntroductionNormothermic ex vivo liver perfusion (NEVLP) is an organ preservation method that allows liver graft functional assessment prior to transplantation. One key component of normothermic perfusion solution is an oxygen carrier to provide oxygen to the liver to sustain metabolic activities. Oxygen carriers such as red blood cells (RBCs) or hemoglobin-based oxygen carriers have an unknown effect on the liver-resident immune cells during NEVLP. In this study, we assessed the effects of different oxygen carriers on the phenotype and function of liver-resident immune cells.MethodsAdult Lewis rat livers underwent NEVLP using three different oxygen carriers: human packed RBCs (pRBCs), rat pRBCs, or Oxyglobin (a synthetic hemoglobin-based oxygen carrier). Hourly perfusate samples were collected for downstream analysis, and livers were digested to isolate immune cells. The concentration of common cytokines was measured in the perfusate, and the immune cells underwent phenotypic characterization with flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The stimulatory function of the liver-resident immune cells was assessed using mixed lymphocyte reactions.ResultsThere were no differences in liver function, liver damage, or histology between the three oxygen carriers. qRT-PCR revealed that the gene expression of nuclear factor κ light chain enhancer of activated B cells (NF-kB), Interleukin (IL-1β), C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 7 (CCL7), and CD14 was significantly upregulated in the human pRBC group compared with that in the naive, whereas the rat pRBC and Oxyglobin groups were not different from that of naive. Flow cytometry demonstrated that the cell surface expression of the immune co-stimulatory protein, CD86, was significantly higher on liver-resident macrophages and plasmacytoid dendritic cells perfused with human pRBC compared to Oxyglobin. Mixed lymphocyte reactions revealed increased allogeneic T-cell proliferation in the human and rat pRBC groups compared to that in the Oxyglobin group.ConclusionsLiver-resident immune cells are important mediators of rejection after transplantation. In this study, we show that the oxygen carrier used in NEVLP solutions can affect the phenotype of these liver-resident immune cells. The synthetic hemoglobin-based oxygen carrier, Oxyglobin, showed the least amount of liver-resident immune cell activation and the least amount of allogeneic proliferation when compared to human or rat pRBCs. To mitigate liver-resident immune cell activation during NEVLP (and subsequent transplantation), Oxyglobin may be an optimal oxygen carrier.

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Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation.

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Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation.

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