Name: lentiviral vector-mediated gene therapy of hepatocytes ex vivo for autologous transplantation in swine
Uploaded: 2018-11-04T13:00:00
Description: Watch this Scientific Journal Video about Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine at JoVE.com

The authors thank Duane Meixner for expertise in performing the portal vein injection,&#160;Steve Krage,&#160;Joanne Pederson, and Lori Hillin for support during the surgical procedures. This work was supported by the Children&#8217;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.

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.&#160; Animals are socially housed unless considered incompatible by institutional veterinary care staff.&#160; Animals are fed ...

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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...

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 is easily assayed through the presence of biomarkers often available in the circulation.
HT-1 is an inborn error of metabolism of the liver that results from a defect in fumarylacetoacetate hydrolase (FAH)5, the last enzymatic step in tyrosine metabolism6. FAH deficiency leads to the build up of toxic metabolites in the liver that can cause acute liver failure and death or in the chronic form of the disease can cause cirrhosis and hepatocellular carcinoma. The disease is clinically managed by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), a small molecule inhibitor of an enzyme upstream of FAH in tyrosine metabolism. The disease provides an ideal environment in which to test gene therapy methods, as successful correction of even a small number of hepatocytes will eventually result in the repopulation of the entire liver with corrected cells in both small and large animal models7,8. This occurs because corrected cells have a profound survival advantage over uncorrected cells due to the accumulation of toxic metabolites in the latter. The loss of uncorrected hepatocytes allows for selective expansion of corrected hepatocytes consistent with the regenerative capacity of the liver. Treatment can be easily followed by measuring the decrease in circulating tyrosine and succinylacetone levels following transplantation.
In order to justify the invasive nature of the procedure, which includes a partial hepatectomy, the goal of this approach must be a durable cure. Therefore, replication incompetent lentiviral vectors are used because they will stably integrate into the hepatocyte genome9. ensuring delivery of the corrected gene to all daughter cells as the liver grows and expands to replace the rapid loss of uncorrected cells. This is advantageous over adeno associated viral (AAV) vectors, which primarily exist as episomes that can only be passed to a single daughter cell during mitosis10 thereby losing any effect of the therapy in a matter of weeks.
Although a growing body of literature supports the safety of lentivirus11, concerns over genotoxic events are mitigated by limiting the transduction of host cells to a controlled in vitro environment. Free vector is never systemically introduced to the host when this method is performed, limiting exposure to the hepatocytes that will be re-introduced with autologous transplant via the portal vein.
This report describes the method of the surgical and ex vivo procedures used to isolate hepatocytes for gene therapy ex vivo and subsequent autologous transplantation12 for the treatment of the HT-1 pig8. The full process includes 1) a partial hepatectomy that serves as a source of hepatocytes and a growth stimulus for the host&#39;s liver, 2) isolation of hepatocytes from the excised liver followed by ex vivo gene correction, and finally 3) reintroduction of the corrected hepatocytes back into the host. The method described is applicable to all large animal models with some modification, but only the FAH-deficient pig13 will have the advantage of the selective environment for corrected hepatocytes.

<table border="0" cellpadding="0" cellspacing="0" width="763">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC)</td>
			<td style="width:139px;">Yecuris</td>
			<td style="width:127px;">20-0027</td>
			<td style="width:109px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12 mm Trocar</td>
			<td>Covidien</td>
			<td>B12STS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mm Trocar</td>
			<td>Covidien</td>
			<td>B5SHF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endo Surgical Stapler 60</td>
			<td>Covidien</td>
			<td>EGIA60AMT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endo Surgical Stapler 45</td>
			<td>Covidien</td>
			<td>EGIA45AVM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endo Surgical Stapler 30</td>
			<td>Covidien</td>
			<td>SIG30AVM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endo catch bag</td>
			<td>Covidien</td>
			<td>173050G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0 PDS</td>
			<td>Ethicon</td>
			<td>Z340H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-0 Vicryl</td>
			<td>Ethicon</td>
			<td>J459H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Vicryl</td>
			<td>Ethicon</td>
			<td>J426H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dermabond</td>
			<td>Ethicon</td>
			<td>DNX12</td>
			<td>Sterile Dressing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Williams&#8217;-E Powder&#160;</td>
			<td>Gibco</td>
			<td>ME16060P1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3&#160;</td>
			<td>Sigma Aldrich</td>
			<td>S8875-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES&#160;</td>
			<td>Fisher</td>
			<td>BP310-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep&#160;</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35-011-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl (g/L)</td>
			<td>Sigma Aldrich</td>
			<td>S1679-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl (g/L)</td>
			<td>Sigma Aldrich</td>
			<td>P3911-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA (g/L)</td>
			<td>Oakwood Chemical</td>
			<td>45172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-acetyl-L-cysteine</td>
			<td>Oakwood Chemical</td>
			<td>3631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(N-A-C, g/L)</td>
			<td>Sigma Aldrich</td>
			<td>A9165-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2 2H2O (g/L)</td>
			<td>Sigma Aldrich</td>
			<td>223506-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase D (mg/mL)</td>
			<td>Crescent Chemical</td>
			<td>17456.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dexamethasone</td>
			<td>Fresenius Kabi</td>
			<td>NDC6337</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal Growth Factor</td>
			<td>Gibco</td>
			<td>PHG0314</td>
			<td></td>
		</tr>
	</tbody>
</table>

lentiviral vector-mediated gene therapy of hepatocytes ex vivo for autologous transplantation in swine

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&#39;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.

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 &#62;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...

Watch this Scientific Journal Video about Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine at JoVE.com

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

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.

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

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 ...

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