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Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Published: November 4th, 2018



1Department of Surgery, Mayo Clinic, 2Midwest Fetal Care Center, Children's Hospitals and Clinics of Minnesota, 3Department of Surgery, Johns Hopkins, 4Pediatric Surgical Associates

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector's ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Inborn errors of metabolism of the liver are a family of genetic diseases that collectively affect as many as 1 in 800 live births1. Many of these diseases are single gene defects2 and can be functionally cured by introducing a single corrected copy of the affected gene into a sufficient number of hepatocytes3. The actual percentage of hepatocytes that needs to be corrected varies by the disease4 and is largely dependent on the nature of the protein it encodes, for example, excreted proteins versus cytoplasmic. In most cases, efficacy of any treatment for metabolic disease ....

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All animal procedures were performed in accordance with institutional guidelines and were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) prior to study conduct. Procedures described here were performed on male and female large white farm pigs (50% Landrace/50% Large White genetic background) up to 3 months of age that are deemed healthy and suitable for surgery.  Animals are socially housed unless considered incompatible by institutional veterinary care staff.  Animals are fed .......

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The liver resection and autologous transplantation are represented schematically in Figure 1. In a representative cohort of 5 pigs that underwent hepatic resection, most had yields of >1 x 109 hepatocytes with approximately 80% viability (Table 2), providing plenty of cells for any type of desired manipulations, including gene therapy. Subsequent culture of the non-transplanted portion of prepared hepatocytes from each of those.......

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This report describes an ex vivo autologous gene therapy approach to cure a porcine model of HT-1. It involves a partial hepatectomy, followed by ex vivo hepatocyte isolation and transduction of isolated hepatocytes with lenti virus carrying the corrective transgene. Corrected autologous hepatocytes are then transplanted back to the FAH deficient animal through the portal vein8. Although the method described is applicable to all large animal models with some modification, the FAH.......

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The authors thank Duane Meixner for expertise in performing the portal vein injection, Steve Krage, Joanne Pederson, and Lori Hillin for support during the surgical procedures. This work was supported by the Children’s Hospital of Minnesota Foundation and Regenerative Medicine Minnesota. R.D.H. was funded through an NIH K01 DK106056 award and a Mayo Clinic Center for Regenerative Medicine Career Development Award.


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Name Company Catalog Number Comments
2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) Yecuris 20-0027
12 mm Trocar Covidien B12STS
5 mm Trocar Covidien B5SHF
Endo Surgical Stapler 60 Covidien EGIA60AMT
Endo Surgical Stapler 45 Covidien EGIA45AVM
Endo Surgical Stapler 30 Covidien SIG30AVM
Endo catch bag Covidien 173050G
0 PDS Ethicon Z340H
2-0 Vicryl Ethicon J459H
4-0 Vicryl Ethicon J426H
Dermabond Ethicon DNX12 Sterile Dressing
Williams’-E Powder  Gibco ME16060P1
NaHCO3  Sigma Aldrich S8875-1KG
HEPES  Fisher BP310-1
Pen/Strep  Gibco 15140-122
Fetal Bovine Serum Corning 35-011-CV
NaCl (g/L) Sigma Aldrich S1679-1KG
KCl (g/L) Sigma Aldrich P3911-500G
EGTA (g/L) Oakwood Chemical 45172
N-acetyl-L-cysteine Oakwood Chemical 3631
(N-A-C, g/L) Sigma Aldrich A9165-100G
CaCl2 2H2O (g/L) Sigma Aldrich 223506-500G
Collagenase D (mg/mL) Crescent Chemical 17456.2
Dulbecco's modified eagle medium (DMEM) Corning 15-013-CV
Dexamethasone Fresenius Kabi NDC6337
Epidermal Growth Factor Gibco PHG0314

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