Hepatitis C

We designed a device for the therapy of hepatitis C, composed of microencapsulated mammalian cells that include a genetic bistable toggle switch with a positive feedback loop, where in one state the cells produce interferon alpha (IFN-α) as the antiviral effector and in the second state they produce hepatocyte growth factor (HGF) to promote liver regeneration. A pharmacokinetic model demonstrated that if the device is implanted into the liver it results in higher levels of IFN-α within the liver than systemically. More importantly, this type of application avoids the spikes of high IFN-α concentration that occur in treatment with IFN-α injections. This should decrease the severity of side effects of IFN-α experienced by a high percentage of patients. We estimated, based on the detection of IFN-α produced by HEK293T cells, that sufficient quantities of the therapeutic protein could be produced by the amount of microencapsulated cells feasible in a real therapeutic application.

Figure 1. Therapy of hepatitis C by microencapsulated cells which can be regulated to produce and release therapeutic proteins into the liver tissue.

Figure 2. Scheme of the constructs for the regulated therapy of hepatitis C with interferon alpha (IFN-α) and hepatocyte growth factor (HGF). Each of the therapeutic effector is released in equimolar amount to the autoactivator.

Hepatitis C virus infection

Figure 3. Hepatitis C prevalence

Hepatitis C is an infectious disease caused by the hepatitis C virus (HCV) which primarily infects the liver. Hepatitis C is a serious worldwide health problem with a prevalence of 3% in the world’s population. According to WHO, 170 million individuals are infected with an incidence of 3 to 4 millions of new cases per year. More than 350,000 people die yearly from hepatitis C-related diseases. In the US the HCV has surpassed HIV as a cause of death. Medical care costs associated with treatment of HCV infection are estimated to be more than $600 million per year just in the USA.

HCV accounts for 20% of all cases of acute hepatitis which is usually asymptomatic. In approximately 75% – 85% of patients, HCV persists as a chronic infection, placing infected persons at risk for developing chronic liver disease. The risk of liver cirrhosis after 20 years of persistent hepatitis C infection is approximately 10-15% for men and 1-5% for women. Once cirrhosis is established, the rate of developing liver cancer is 1 to 4% per year (Pawlotsky, 2004). It is the most common indication for orthotopic liver transplantation in the United States.

Hepatitis C virus is a small, spherical, enveloped, single-stranded RNA virus, a member of the Flaviviridae family. Because the HCV RNA-dependent RNA polymerase lacks proofreading capabilities, it generates a large number of mutant viruses known as quasispecies when the virus replication takes place. These HCV quasispecies represent a major challenge to immune-mediated control of HCV and may explain the difficulties in vaccine development.

Current therapy

Patients with an acute hepatitis C virus infection which has not resolved after 2-4 months are treated with standard interferon (IFN) therapy for 6 months. Treatment of chronic HCV infection has two goals: sustained eradication of HCV and prevention of chronic complications. IFN-α has been used since the 1980s in the treatment of chronic hepatitis and still represents an important part of the management of chronic hepatitis C infection. Initial studies used IFN-α monotherapy, but current treatments are a combination therapy consisting of ribavirin and IFN-alpha (Feld and Hoofnagle, 2005). Side effects of treatment are very common, with half of the patients suffering from flu like symptoms and a third experiencing emotional problems. Anxiety, sleep disorders and irritability are frequently observered and can in some cases lead to severe behavioral or psychological disorders. This can in turn lead to the discontinuation of therapy.

Implementation of the microencapsulated synthetic cellular device for the therapy of hepatitis C

With our biological delivery system we aim to overcome some of the adverse effects of the IFN therapy and make the therapy more efficient and affordable. Because of the local (intrahepatic/intraperitoneal) production of IFN-α from the cell-delivery system, a lower systemic concentration is expected, which could diminish systemic side effects. Furthermore, because repetitive subcutaneous application would no longer be needed, the quality of life of patients would improve as well as compliance to the therapy.

In addition to antiviral activity the design of our system allows us to include additional effector proteins in the therapy. Since the advanced stages of hepatitis seriously damage the liver, we decided to include a therapeutic drug to improve liver regeneration. Hepatocyte growth factor (HGF) is a paracrine cellular growth, motility and morphogenic factor secreted by mesenchymal cells. It targets and acts primarily upon epithelial and endothelial cells, but also acts on haematopoietic progenitor cells. It has been shown to have a major role in adult organ regeneration and in wound healing. HGF induces proliferation and regeneration of hepatocytes, and its application by gene therapy in several preclinical studies has inhibited fibrogenesis and hepatocyte apoptosis and produced the complete resolution of fibrosis in the cirrhotic liver (Ueki et al., 1999). The inclusion of HGF to the interferon therapy of hepatitis C could improve liver regeneration as HGF also promotes differentiation of stem cells into hepatocytes (Oyagi et al., 2006, Nakamure et al., 2011).

We adapted our therapeutic device for the therapy of HCV infection to include the coding regions for IFN-α2 and HGF coupled via a t2a peptide sequence to the activators TALA and TALB of our bistable toggle switch with a positive feedback loop (Figure 2). This arrangement allows initial production of IFN-α to treat the viral infection, and an induced switch to the second state after the virus has been cleared allows improved liver regeneration with HGF production.

Results

Pharmacokinetic modeling of the distribution of IFN-α in the therapy

Consultation with Peter Popovič, MD, MSc, an interventional radiologist, suggested that microcapsules could be applied to the liver artery via a catheter to distribute production of IFN-α and later HGH throughout the liver.

A pharmacokinetic model of the localized production of IFN-α in the liver was performed based on parameters from the available literature. The model is described in details in the Modeling section. Modeling demonstrated that a therapy which consists of injecting IFN-α three times a week, exhibits peaks and dips in concentration of the drug in the liver as well as in plasma. The model of our new approach demonstrates that the time course of IFN-α released by the implanted microcapsules is very stable. However the systemic level of IFN-α is not much lower than the concentration in the liver (70% of the latter) because the liver is an organ that is strongly perfused with blood. This can be observed on the animation shown bellow (Figure 4).

Figure 4. Distribution of IFN-α in different organs of the body based on the pharmacokinetic model for the therapy of hepatitis C.

Production of IFN-α

We tested the biological activity of IFN-α produced by HEK293T cells from our construct to get an estimate if the production level could reach a sufficient level. Stably transfected therapeutic cells could be selected for high production for the real therapy. Type I interferons (IFN-α and β) mediate signaling through the IFNAR receptor, the STAT1 and STAT2 components of the JAK/STAT-signal transduction pathways and finally, the interferon stimulated response element (ISRE). We used a STAT1/STAT2-responsive luciferase construct that encodes the firefly luciferase reporter gene under the control of a minimal promoter and tandem repeats of ISRE.

We designed the experiment as a co-culture of HEK293T cells transfected with either the IFN-α encoding plasmid or an empty vector for control and HEK293T cells transfected with the reporter vector. Additionally, we performed a co-transfection experiment, where HEK293T cells were transfected with both the reporter and the IFN-α encoding plasmids (Figure 5). As a positive control, we used the response of the same reporter to recombinant IFN-β.

Figure 5. Expressed and secreted IFN-α induces expression of an ISRE-dependent reporter. A co-culture of HEK293T cells transfected with either the IFN-α producing plasmid under the control of constitutive promoter or an empty vector and HEK293T cells transfected with firefly luciferase reporter with ISRE was prepared. Additionally, HEK293T cells were cotransfected with both the reporter and the IFN-α producing or control plasmids. The response of the same reporter to recombinant IFN-β was used as a positive control. After 24 hours of incubation, a dual luciferase reporter assay was performed.

To assess the required quantity of microcapsules to achieve the therapeutic amount of IFN-α, we performed an enzyme-linked immunosorbent assay (ELISA) of the IFN-α production of HEK293T cells. We determined that the average production of IFN-α in a single cell in 24 hours is approximately 4,6 *10^-9 µg . Pharmacokinetic modeling predicted the required production of interferon to achieve the theraopeutic concentration in the liver to be around 3 µg per day. Considering that one capsule contains approximately 3000 cells, we would have to implant 230,000 capsules into the liver. This number of capsules occupies a volume of roughly 15 mL. We believe that this is a feasible amount for the delivery of synthetic devices into the body. Furthermore, we believe that production of the protein could be significantly increased as HEK293 cells can produce as much as 300 mg of IFN-α per liter of serum-free medium (Loignon et al., 2008).

References

Feld, J.J. and Hoofnagle, H. (2005) Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature 436, 967-72.

Loignon M, Perret S, Kelly J, Boulais D, Cass B, Bisson L, Afkhamizarreh F, Durocher Y. (2008) Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells. BMC Biotechnol. 8, 65.

Nakamura, T., Sakai, K., Nakamura, T. and Matsumoto, K. (2011) Hepatocyte growth factor twenty years on: Much more than a growth factor. J Gastroenterol. Hepatol. 26, 188-202.

Oyagi, S., Hirose, M., Kojima, M., Okuyama, M., Kawase, M., Nakamura, T., Ohgushi, H. and Yagi, K. (2006) Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CCl4-injured rats. J Hepatol. 44, 742-8.

Pawlotsky, J.M. (2004) Pathophysiology of hepatitis C virus infection and related liver disease. Trends in Microbiology. 12, 96-102.

Ueki, T., Kaneda, Y., Tsutsui, H., Nakanishi, K., Sawa, Y., Morishita, R., Matsumoto, K., Nakamura, T., Takahashi, H., Okamoto, E. and Fujimoto, J. (1999) Hepatocyte growth factor gene therapy of liver cirrhosis in rats. Nat. Medicine 5, 226-30.

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