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 appropriate levels of chow twice daily and observed for clinical signs by care staff at least once daily.
1. Preparation for Surgery
<ol>
	<li>Administer 5 mg/kg telazol and 2 mg/kg xylazine intramuscularly to induce sedation. Administer buprenorphine SR subcutaneously at a dose of 0.18 mg/kg for post-operative analgesia (anticipated analgesia from this dose will be up to 72 h). Manually check the animal for responses to verify sedation.</li>
	<li>Once sedated, insert an intravenous catheter into a peripheral ear vein for pharmacologic access.</li>
	<li>Administer intravenous 25 mg/kg cefazolin and intramuscular 5 mg/kg of ceftiofur pre-operatively.
	<ol>
		<li>Administer normal saline intravenously to keep blood pressure and heart rate within normal physiologic limits throughout the procedure.</li>
	</ol>
	</li>
	<li>Place an orogastric tube to decompress the stomach. Perform endotracheal intubation with an appropriately sized endotracheal tube, verify proper placement by compressing the chest manually to detect exhalation, and then mechanically ventilate the animal with 1&#8211;3% isoflurane.</li>
	<li>Place electrocardiogram (ECG) leads, blood pressure cuff, temperature probe, and pulse-oximeter to monitor and record vital signs. Place the animal in a supine position, scrub the abdomen from rib cage to pelvis with betadine, and drape sterilely.</li>
	<li>Ventilate subject with 1-3% isoflurane during the procedure to maintain a surgical plane of anesthesia. Continue to monitor vital signs for changes and adjust isoflurane accordingly to maintain heart rate&#160;at pre-incision levels If blood pressure drops dramatically, reduce isoflurane and initiate institutionally-approved agents to restore vitality, such as intravenous epinephrine.
	<ol>
		<li>Record heart rate, respiration rate, blood pressure, and body temperature on a surgical procedure record to track and maintain animal welfare as per institution-specific policy to ensure compliance with large animal welfare standards.</li>
	</ol>
	</li>
</ol>
2. Laparoscopic Partial Hepatectomy
<ol>
	<li>Perform initial port site entry using an open Hassen technique cephalad to the umbilicus. When peritoneum is visualized, safely introduce a 12 mm trocar into the abdominal cavity. Pass a 5 mm, 30&#176;, scope through this port.</li>
	<li>Insufflate the abdomen with CO2 to 15 mmHg. Under direct visualization with the laparoscopic camera, place two additional 5 mm ports triangulating on the left lateral lobe of the liver. Exact placement of the ports will depend on size and anatomy of the animal.
	<ol>
		<li>At this point, place the laparoscope in one of the 5 mm ports.</li>
	</ol>
	</li>
	<li>Identify the left lateral segment of the liver at the point of the major fissure of left lobe. This fissure separates the medical and left lateral segments. Secure the portal structures along with the hepatic vein along this fissure using a surgical stapler (see Table of Materials). Transect the parenchyma through this fissure using sequential firings from the stapler using 60, 45, or 30 mm long vascular loads.
	<ol>
		<li>When parenchymal resection is complete, assess the remnant liver to ensure adequate hemostasis. Control any bleeding from the parenchyma with cautery or suture.</li>
	</ol>
	</li>
	<li>Retrieve the liver section using endoscopic graspers and an endoscopic tissue retrieval bag.</li>
	<li>Remove the ports and close the 12 mm port incision in three layers with interrupted size 0 suture for the midline fascia, a running 2-0 suture for the deep dermal layer and a running 4-0 suture for the subcuticular layer. The 5 mm ports may be closed in one layer with 2-0.</li>
	<li>Place a sterile dressing on the incisions (see Table of Materials).</li>
	<li>When cells will be ready in less than 4 hours, maintain the animals under general anesthesia post-operatively until the time of autotransplantation.</li>
	<li>Alternatively, if cell manipulations require longer periods (such as to allow formation of hepatocyte spheroids or longer transduction/selection periods based on individual applications of this method) allow the animal to recover from anesthesia and repeat the anesthetic induction steps prior to autotransplantation when the cells are ready.</li>
</ol>
3. Hepatocyte Isolation
<ol>
	<li>Connect a peristaltic pump with perfusion set to deliver warm dispersion solutions (maintained in 43 &#176;C water bath)&#160; to catheters to be placed in the large exposed vessels of the liver section, such that solution temperature is 38 &#176;C upon introduction to the tissue. All tubing, equipment, and solutions must be maintained sterile to ensure the cells are suitable for transplantation.
	<ol>
		<li>Prime the pump and tubing with Per II up to a stopcock positioned to switch between Per I and Per II delivery to the tissue. Switch the stopcock to the Per I position and prime the rest of the tubing set, filling an in-line bubble trap completely to prevent introducing air bubbles into the tissue.</li>
	</ol>
	</li>
	<li>Prepare a clean transverse cut perpendicular to the hepatic circulation to reveal cross sections of portal vein branches and hepatic veins for catheterization.</li>
	<li>Catheterize the available exposed veins with a snug-fitting catheter to avoid leakage of the perfusate. Cannulate at least 1 portal and 1 hepatic vein to ensure adequate perfusion of the tissue.
	<ol>
		<li>Ensure that the catheter(s) are primed/free of air before placing the catheter into a vein.</li>
	</ol>
	</li>
	<li>Perfuse with warm Per I at 100 mL/min, moving the outlet tube from vein to vein every 30 seconds to 1 min. Cycle through all available veins for 15&#8211;20 min. During Per I infusion, set an evacuation tube to empty the procedure tray into a waste container under vacuum.</li>
	<li>Switch to Per II at 100 mL/min, cycling through the veins as with Per I, for a total of 30 min. During Per II, use a return pump to recycle Per II back to the reservoir for re-administration to conserve preparation of the active enzyme. Set the return pump to less than the outflow pump (i.e., 94 mL/min), being careful not to return excessive air to the source bottle.
	<ol>
		<li>If the liver capsule breaks, this time can be reduced.</li>
		<li>If the liver is not fully digested (does not remain dimpled with gentle focal pressure) an additional 5 min of perfusion can be performed.</li>
	</ol>
	</li>
	<li>Occasionally (approximately every 5 minutes) document surface temperature of the bubble trap and/or liver to ensure the hepatocytes are not getting cold. If the liver is getting cold (&#60;35 &#176;C) increase the heat of the water bath to restore the liver temperature closer to 38 &#176;C. The liver should blanche as the perfusion proceeds.</li>
	<li>Remove liver to sterile plate or Petri dish for transport to cell culture hood. Cover sterile field with a sterile drape to maintain integrity during the hepatocyte processing.</li>
	<li>Expose the liver section from sterile drape and submerge the liver with cold hepatocyte wash media (HWM). Disrupt the capsule by dragging scissors across the top. Wearing sterile gloves, gently massage the liver to release the hepatocytes into the medium.</li>
	<li>Filter the HWM containing hepatocytes through sterile gauze into large (~200 mL) centrifuge bottles. Wash the hepatocytes through the following procedure in triplicate, combining the cell pellet from the multiple bottles after the first resuspension (as applicable):
	<ol>
		<li>Centrifuge at 50 x g, 5 min, 4 &#176;C.</li>
		<li>Aspirate and resuspend in 150 mL of HWM.</li>
		<li>Leave the pellet in HWM after the 3rd wash.</li>
	</ol>
	</li>
	<li>Count cell concentration using a hemocytometer or other device, as available. These cells are now ready for ex vivo manipulation of interest, including gene therapy, cell sorting, or other specialization prior to autotransplantation. Final volume of HWM can be adjusted to target a specific concentration (cells/mL).</li>
</ol>
4. Hepatocyte Transduction
<ol>
	<li>Resuspend hepatocytes in media (Dulbecco&#8217;s modified eagle medium [DMEM], 10% fetal bovine serum [FBS], Penicillin-Streptomycin, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], dexamethasone, epidermal growth factor [EGF], and NTBC) at 1&#8211;2 million cells per mL in a conical tube on ice.</li>
	<li>Thaw lentiviral vector at room temperature and add to conical tube on ice at target 20 multiplicity of infection (MOI) to bind the cells. Rotate cells at 4 &#176;C at 10 rpm for 90 min to bind the viral vector to the cell surface.</li>
	<li>Transfer tubes to 37 &#176;C for 30&#8211;40 min. Invert to mix every 5 minutes to facilitate the vector transducing the bound cells.</li>
	<li>Centrifuge cells at 50 x g at 4 &#176;C for 4 min. Resuspend cell pellet in saline at desired concentration on ice. Repeat this wash to ensure removal of any unbound vector from the preparation.</li>
	<li>If possible, plate sufficient aliquots of cells (i.e., 500,000 cells per well in a 6 well cell culture plate) to check for viability, adhesion, transduction efficiency, etc. 
	NOTE: It is often not possible to assess these parameters prior to autotransplantation, especially for same-day procedures. For example, the procedure described herein employed an untagged Fah cDNA under the control of the alpha-1 antitrypsin liver-specific promoter, which was not readily assayable in the hepatocytes prior to transplantation. However, these plated cells can be assayed subsequent to the autotransplantation as an indicator of the success of transduction (i.e., via Western blotting) or a predictor of the general success of the procedure.</li>
</ol>
5. Hepatocyte Transplantation
<ol>
	<li>Identify the portal vein via ultrasound using a 2 to 5 MHz transducer. Direct an 18 G, 5 inch needle towards the main portal vein proximal to its bifurcation.</li>
	<li>Begin slow manual infusion of up 1 x 109 hepatocytes (approximately 10 g) using a syringe. Monitor portal pressures during transplantation using an infusion catheter.
	<ol>
		<li>If portal pressures increase more than 8 mmHg above baseline, pause infusion and allow the pressure to return to baseline levels.</li>
		<li>If portal pressures do not return to baseline after pausing infusion for five minutes, discontinue the infusion.</li>
	</ol>
	</li>
	<li>After transplantation and catheter removal, use the ultrasound to evaluate for the presence of thrombotic events and forward flow in the portal vein.</li>
</ol>
6. Postoperative Recovery and Maintenance
<ol>
	<li>Move subject to a proper recovery area and observe until the animal is fully ambulatory, monitoring heart rate, oxygen saturation and body temperature every 15&#8211;30 min. Maintain body temperature with warm blankets or an air-heated wrap, as needed.</li>
	<li>Administer 1 mg/kg omeprazole by mouth daily (through the end of the study) to reduce the chances of gastric ulceration that can occur with bouts of inappetence.</li>
	<li>Postoperatively, the animal is monitored closely by lab and veterinary staff and typically does not require additional pain medication separate from the pre-operative buprenorphine dose.&#160; If signs of distress/pain are observed by qualified veterinary care staff (incision guarding, inappetence, lethargy), anti-inflammatory agents (i.e., Carprofen) may be administered.</li>
</ol>
7. Recipes
<ol>
	<li>See Table 1 for recipes used in this protocol.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>HWM</td>
			<td>Reagent</td>
			<td>Per I (10x)</td>
			<td>Per II (10x)</td>
		</tr>
		<tr>
			<td>Williams&#8217;-E Powder (g/L)</td>
			<td>10.8</td>
			<td>NaCl (g/L)</td>
			<td>83</td>
			<td>39</td>
		</tr>
		<tr>
			<td>NaHCO3 (g/L)</td>
			<td>2.2</td>
			<td>KCl (g/L)</td>
			<td>5</td>
			<td>5</td>
		</tr>
		<tr>
			<td>HEPES (g/L)</td>
			<td>2.6</td>
			<td>HEPES (g/L)</td>
			<td>24</td>
			<td>240</td>
		</tr>
		<tr>
			<td>Pen/Strep (100x, mL/L)</td>
			<td>10</td>
			<td>EGTA (g/L)</td>
			<td>9.5</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (mL/L)</td>
			<td>100</td>
			<td>N-acetyl-L-cysteine</td>
			<td rowspan="2">8</td>
			<td rowspan="2">8</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.3</td>
			<td>(N-A-C, g/L)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Nitroglycerin (mL/L)</td>
			<td>5</td>
			<td>5</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>CaCl2 2H2O (g/L)</td>
			<td>-</td>
			<td>7</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Collagenase D (mg/mL)</td>
			<td>-</td>
			<td>0.2</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>pH</td>
			<td>7.4</td>
			<td>7.6</td>
		</tr>
	</tbody>
</table>
Table 1: Recipes for Solutions Used in Hepatocyte Isolation from Liver Sections.

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+for+inborn+errors+of+metabolism:+advancing+towards+the+clinic.">Genome editing for inborn errors of metabolism: advancing towards the clinic.</a> BMC Medicine. 15 (1), 43 (2017).</li><li>Brunetti-Pierri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+inborn+errors+of+liver+metabolism:+progress+towards+clinical+applications.">Gene therapy for inborn errors of liver metabolism: progress towards clinical applications.</a> Italian Journal of Pediatrics. 34 (1), 2 (2008).</li><li>Carlson, D. 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The authors have nothing to disclose.

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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8.0K Views.  Mayo Clinic. 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.

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

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery.  Fresh patient tissue obtained at the time of surgery ...

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...