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

Ex vivo gene therapy and autologous cell transplantation for the treatment of inborn errors of metabolism in the liver is an important alternative to the therapy for these disorders. And allows us to not only treat these disorders, but figure out the biomass necessary to be able to achieve a therapeutic result. This approach also avoids the significant consequences of immunosuppression and other consequences with different types of therapies.

In the current context, we're using this approach to treat hereditary tyrosinemia type 1. However, the idea is that this will be a platform approach to treat a multitude of different liver diseases with minimal alteration. It's important to be able to visualize this technique because there's some variability when it comes to visualizing the efficiency of the perfusion, which is essential to getting viable hepatocytes for the transplantation.

Surgical procedure performed by Robert Kaiser and Joseph Lillegard. Cell isolation performed by Zeji Du, Bruce Amiot. UAU cell purification and ex vivo transduction of the cells was done by Caitlin VanLith.

Preparation of the animals and the OR prep was done by Kari Allen and Laurie Hillin. To perform the initial port site entry, perform an open Hasson technique cephalad to the umbilicus of an anesthetized, up to three-month-old, large, white farm pig. Introducing a 12 millimeter trocar into the abdominal cavity, where the peritoneum is visible.

When the trocar is in place, pass a 5 millimeter 30 degree scope through the entry port and insufflate the abdomen with carbon dioxide to 15 millimeters of mercury. Under direct visualization with the laparoscopic camera, place 2 additional 5 millimeter ports, triangulating on the left lateral lobe of the liver. And place the laparoscope in one the new ports.

Identify the left lateral segment of the liver at the point of the major fissure of the left lobe that separates the medial and left lateral segments. And use a surgical stapler to secure the vascular structures and the hepatic vein along this fissure. Transect the parenchyma through this fissure using sequential firings and 60, 45, or 30 millimeter long vascular loads.

When the resection is complete, assess the remnant liver to ensure an adequate hemostasis and use endoscopic graspers and an endoscopic tissue retrieval bag to retrieve the liver section. Now, remove the ports and use an interrupted size zero 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, to close the 12 millimeter port incision in the three layers. Then close the five millimeter ports in one layer with 2-0 sutures. And place a sterile dressing on the incisions.

For hepatocyte isolation, first connect a peristaltic pump set to deliver the dispersion solutions, maintained in a 43 degree Celsius water bath, to the catheters to be placed in the large, exposed vessels of the liver section. Prime the pump and tubing with the perfusion two solution, up to a stop cock position to switch between the perfusion one and perfusion two solution delivery to the tissue. And switch the stop cock to the perfusion one position.

Then prime the rest of the tubing set. Completely filling an inline bubble trap to prevent introducing air bubbles into the tissue. Next, make a clean transverse incision, perpendicular to the hepatic circulation to reveal cross-sections of the portal vein branches and the hepatic veins for catheterization.

And use a snug fitting catheter to catheterize the available, exposed veins. When the catheters are in place, perfuse the resected liver tissue with warm perfusion one solution at a 100 millimeter per minute flow rate. Moving the outlet tube from vein to vein every 30 to 60 seconds.

And using an evacuation tube to remove excess buffer from the tissue tray. After cycling through all of the available veins for 15 to 20 minutes, switch to perfusion two solution under the same conditions. Using a return pump to recycle the perfusion two solution back to the reservoir, at a slower flow rate, for re-administration into the tissue.

Check the surface temperature of the bubble trap and, or the liver about every five minutes to confirm that the hepatocytes are not getting cold. When the liver blanches after about 30 minutes of perfusion, transfer the tissue to a sterile container, wrapped with a sterile drap, and place the container into a cell culture hood to maintain sterility during the hepatocyte processing. The collagenase isolation of the hepatocytes and cell disassociation needs to be done completely so you don't get clumping of the cells which decreases the viability of the cells and the transduction frequency.

Submerge the liver with hepatocyte wash medium and drag scissors across the top of the tissue to disrupt the liver capsule. Wearing sterile gloves, gently massage the liver to release the hepatocytes into the medium. And filter the tissue supernatant, through sterile gauze, into 200 milliliters centrifuge bottles.

Collect the filtered hepatocytes by centrifugation. Pooling the cells in 150 milliliters of fresh hepatocyte wash medium into a single 200 milliliter bottle for 2 additional washes. And counting the cells after the third centrifugation.

Then, dilute the cells to 1 times 10 to the ninth hepatocytes per milliliter saline concentration. For autotransplantation of the isolated hepatocytes, use a two-five megahertz transducer to identify the portal vein via ultrasound and direct a five-inch, 18-gauge needle toward the main portal vein, approximal to it's bifurcation. Load the cells into a 60 milliliter syringe, according to the total cell volume and attach the syringe to the needle, taking care not to introduce bubbles.

Next, slowly depress the syringe plunger to manually infuse up to 1 times 10 to the ninth hepatocytes through the portal vein using an infusion catheter to monitor the poral pressure during the transplantation. When all of the cells have been delivered, remove the catheter and monitor the portal vein for the presence of thrombotic events and forward flow by ultrasound. In a representative cohort of five pigs, that underwent hepatic resection, most had yields of greater than 10 to the ninth hepatocytes with approximately 80%viability.

Providing a sufficient number of cells for any type of desired manipulation, including gene therapy. Subsequent culture of the non-transplanted portion of these prepared hepatocytes, demonstrated a good viability and adhesion with a typical hepatocyte morphology 46 hours after their transduction and initial plating. An initial engraftment will vary by the number of cells reintroduced during the transplantation.

These representative biopsies demonstrate a timeline of gene-corrected cell expansion. Unique to diseases, such as hereditary tyrosinemia type 1, that enjoy a positive selected pressure for healthy cells. This expansion is typically complete 12 months after transplantation.

Once a transplanted fumarylacetoacetate hydrolase knockout animal has achieved about 20%re-population of the corrected hepatocytes within the liver, the tyrosine levels will normalize compared to wild-type animals. While uncorrected animals exhibit a significant elevation in both alkaline phosphatase and aspartate transaminase compared to wild-type animals, ex vivo gene therapy returns these serum enzymes levels to normal. Further, the general liver metabolic health is disrupted in untreated knockout pigs, as indicated by elevations in the circulating ammonia that are corrected after re-population with the treated hepatocytes.

It's important to remember with using this technique to handle and manipulate the tissue carefully and as little as possible. Increased handling or manipulation of the tissue, leads to decrease viability of the cells and decrease yield. This approach can also be applicable for people who are interested in studying bow distribution and graph inefficiencies in labeling techniques, using other techniques outside of viral transduction protocols.

Don't forget that working with viral vectors can be hazardous, so following proper bio safety procedures, as well as wearing personal protective equipment is necessary when using these techniques.