Kathryn L. Nash

Hepatocyte-specific gene expression from integrated lentiviral vectors

For many applications, efficient gene therapy will require long‐term, organ‐specific therapeutic gene expression. Lentiviral vectors based on HIV‐1 are promising gene delivery vehicles due to their ability to integrate transgenes into non‐dividing cells. Many experimental vectors express transgenes under the control of the cytomegalovirus (CMV) immediate‐early gene promoter. Although this promoter directs strong gene expression in vitro, it may be shut off rapidly in vivo. This study explores the potential of HIV‐1‐based vectors to transduce hepatocytes and compares gene expression from different promoters in integrated vectors.

Age-related macular degeneration and modification of systemic complement factor H production through liver transplantation.

PURPOSE To investigate whether modification of liver complement factor H (CFH) production, by alteration of liver CFH Y402H genotype through liver transplantation (LT), influences the development of age-related macular degeneration (AMD). DESIGN Multicenter, cross-sectional study. PARTICIPANTS We recruited 223 Western European patients ≥ 55 years old who had undergone LT ≥ 5 years previously. METHODS We determined AMD status using a standard grading system. Recipient CFH Y402H genotype was obtained from DNA extracted from recipient blood samples. Donor CFH Y402H genotype was inferred from recipient plasma CFH Y402H protein allotype, measured using enzyme-linked immunosorbent assays. This approach was verified by genotyping donor tissue from a subgroup of patients. Systemic complement activity was ascertained by measuring levels of plasma complement proteins using an enzyme-linked immunosorbent assay, including substrates (C3, C4), activation products (C3a, C4a, and terminal complement complex), and regulators (total CFH, C1 inhibitor). MAIN OUTCOME MEASURES We evaluated AMD status and recipient and donor CFH Y402H genotype. RESULTS In LT patients, AMD was associated with recipient CFH Y402H genotype (P = 0.036; odds ratio [OR], 1.6; 95% confidence interval [CI], 1.0-2.4) but not with donor CFH Y402H genotype (P = 0.626), after controlling for age, sex, smoking status, and body mass index. Recipient plasma CFH Y402H protein allotype predicted donor CFH Y402H genotype with 100% accuracy (n = 49). Plasma complement protein or activation product levels were similar in LT patients with and without AMD. Compared with previously reported prevalence figures (Rotterdam Study), LT patients demonstrated a high prevalence of both AMD (64.6% vs 37.1%; OR, 3.09; P<0.001) and the CFH Y402H sequence variation (41.9% vs 36.2%; OR, 1.27; P = 0.014). CONCLUSIONS Presence of AMD is not associated with modification of hepatic CFH production. In addition, AMD is not associated with systemic complement activity in LT patients. These findings suggest that local intraocular complement activity is of greater importance in AMD pathogenesis. The high AMD prevalence observed in LT patients may be associated with the increased frequency of the CFH Y402H sequence variation. FINANCIAL DISCLOSURE(S) The authors have no proprietary or commercial interest in any materials discussed in this article.

Inhibition of hepatitis B virus by lentiviral vector delivered antisense RNA and hammerhead ribozymes.

Summary.  Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. Current treatments are limited and may be ineffective. Nucleic acid‐mediated targeting of viral mRNA is an attractive and specific approach for viral infection and lentiviral vectors provide a means to express antisense sequences or ribozymes stably in target cells permitting continuous production within that cell and its progeny. To demonstrate long‐term gene expression by lentiviral vectors in hepatocytes and to introduce lentiviral vectors expressing anti‐HBV genes to assess their effect against HBV, lentiviral vectors expressing a reporter gene were assessed for longevity of gene expression in hepatocytes in vitro. Hammerhead ribozymes and antisense sequences targeting the HBV encapsidation signal (ε), X or surface antigen on mRNAs were cloned into lentiviral vectors and used to transduce HBV expressing hepatocytes where the effect on HBV mRNA level was assessed using ribonuclease protection. Gene expression in hepatocytes from integrated vectors continued for over 4 months without selection. Antisense RNA targeting HBs mRNA reduced this transcript, whilst antisense RNA to HBX mRNA was ineffective. Sense RNAs corresponding to ɛ and HBX mRNA also reduced HBV mRNA levels. Ribozymes targeting HBs and HBX mRNA effectively reduced HBV mRNA levels compared with inactive constructs indicating their effect to be enzymatic rather than antisense. Lentiviral vectors can produce long‐term gene expression in hepatocytes and thus permit prolonged expression of antiviral genes targeting the HBV encapsidation signal, surface and X mRNAs as treatments for chronic HBV infection.

The case for combination antiviral therapy for chronic hepatitis B virus infection

The treatment of hepatitis B virus (HBV) infection has been revolutionised in the past decade by the increased availability of effective antiviral agents. Many studies have shown the benefits of single agent therapy, but there is an alarming and rising rate of viral resistance, and clear evidence that viruses that harbour resistant mutations can cause liver disease and death. Current national guidelines for the treatment of HBV recommend a programme that starts with monotherapy, followed by sequential monotherapy or add-on therapy for those infections in which mutations have arisen. Very few studies starting with combination therapy have been undertaken, so there is little evidence of the clinical benefit of this approach to treatment. The studies that have been done have been short term and have concentrated on clinical parameters rather than virological resistance, which is likely to be the key determinant in the longer term. We argue that we should not wait for the evidence to use combination therapy for the treatment of HBV, since such trials may never be done and it would take several years for a benefit to become apparent. In the meantime, multidrug-resistant strains continue to hinder HBV control.

Is cyclosporin the immunosuppressant of choice for liver transplantation for erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) occurs as a result of ferrochelatase deficiency, the final enzyme of the haem biosynthetic pathway, leading to protoporphyrinaemia, which causes skin photosensitivity; 15–20% of patients also develop hepatic dysfunction including gall stones, cirrhosis and in a minority, fulminant hepatic failure [1,2]. Patients with severe liver disease may be candidates for liver transplantation, although it is not curative as excessive protoporphyrin (produced mainly in the bone marrow) is reported to cause graft injury [3,4]. We describe a patient with EPP who underwent liver transplantation and in whom the graft revealed features of severe injury at a very early stage as a consequence of protoporphyric hepatopathy. Switching to an alternative calcineurin inhibitor improved porphyrin metabolism and clinical status. A 34-year-old man presented with sudden onset of abdominal pain, jaundice, pruritus and increased photosensitivity. Protoporphyria had been diagnosed at the age of 6 months and he had, from that point onwards, avoided sunlight. On examination, he had fragile skin, jaundice and hepatosplenomegaly. Results of serum tests of liver biochemistry and porphyrin concentrations are shown in Table 1. There was no evidence of cholelithiasis or haemolysis. Decompensated protoporphyric hepatopathy was diagnosed and attempts were made to reduce protoporphyrinaemia including hyper-transfusion, cholestyramine, ursodeoxycholic acid and antioxidant treatment with vitamins C and E and cysteine. His condition continued to deteriorate with the onset of liver failure and he underwent liver transplantation. He received standard immunosuppression with tacrolimus (trough level 10–15 lg/l), prednisolone and azathioprine. The early postoperative course was complicated by two episodes of biopsy-proven acute cellular rejection, which resolved with pulsed methylprednisolone and a biliary stricture that was successfully dilated. The measures instituted to reduce protoporphyrinaemia were continued. He was maintained on tacrolimus (trough level 10–15 lg/l), azathioprine 50 mg/day and a reducing course of prednisolone along with valganciclovir as prophylaxis against cytomegalovirus (CMV) infection, nystatin and ranitidine. A liver biopsy performed 4 months after transplantation because of deranged graft function revealed features characteristic of protoporphyric hepatopathy with accumulation of protoporphyrins, fibrous expansion of portal tracts, cholestasis and hepatocyte degeneration and grade II siderosis, consistent with transfusion. No other cause of graft injury was present. Jaundice and photosensitivity continued to worsen in association with increasing porphyrinaemia (Table 1). Cyclosporin was substituted for tacrolimus and his clinical condition, photosensitivity and porphyrin biochemistry improved rapidly (Table 1). Three years later, blood porphyrin concentrations and