Gene and cell therapy in South Africa: Current status and future prospects.

South Africa has a high disease burden resulting from communicable and non-communicable diseases. Current therapeutic interventions rarely result in a cure and the associated lifelong treatment places a considerable strain on an overburdened health sector. Gene and cell therapies present novel alternatives to disease management, offering the promise of a single treatment and a lifelong cure. Although challenges remain, investment in the field has started to bear fruit, with a number of gene and cell therapeutics reaching the market in the past decade. To take full advantage of these developments, it is important that a proactive approach to nurturing appropriate human and material resources is adopted in the country.

Gene and cell therapy in South Africa: current status and future prospects

South Africa has a high disease burden resulting from communicable and non-communicable diseases. Current therapeutic interventions rarely result in a cure and the associated lifelong treatment places a considerable strain on an overburdened health sector. Gene and cell therapies present novel alternatives to disease management, offering the promise of a single treatment and a lifelong cure. Although challenges remain, investment in the field has started to bear fruit, with a number of gene and cell therapeutics reaching the market in the past decade. To take full advantage of these developments, it is important that a proactive approach to nurturing appropriate human and material resources is adopted in the country

The state of gene therapy research in Africa, its significance and implications for the future.

Gene therapy has made impressive recent progress and has potential for treating a wide range of diseases, many of which are important to Africa. However, as a result of lack of direct public funding and skilled personnel, direct research on gene therapy in Africa is currently limited and resources to support the endeavor are modest. A strength of the technology is that it is based on principles of rational design, and the tools of gene therapy are now highly versatile. For example gene silencing and gene editing may be used to disable viral genes for therapeutic purposes. Gene therapy may thus lead to cure from infections with HIV-1, hepatitis B virus and Ebola virus, which are of significant public health importance in Africa. Although enthusiasm for gene therapy is justified, significant challenges to implementing the technology remain. These include ensuring efficient delivery of therapeutic nucleic acids to target cells, limiting unintended effects, cost and complexity of treatment regimens. In addition, implementation of effective legislation that will govern gene therapy research will be a challenge. Nevertheless, it is an exciting prospect that gene therapy should soon reach the mainstream of medical management. Participation of African researchers in the exciting developments is currently limited, but their involvement is important to address health problems, develop capacity and enhance economic progress of the continent.

Sustained inhibition of hepatitis B virus replication in vivo using RNAi-activating lentiviruses.

Chronic infection with hepatitis B virus (HBV) puts individuals at high risk for complicating cirrhosis and liver cancer, but available treatment to counter the virus rarely eliminates infection. Although harnessing RNA interference (RNAi) to silence HBV genes has shown the potential, achieving efficient and durable silencing of viral genes remains an important goal. Here we report on the propagation of lentiviral vectors (LVs) that successfully deliver HBV-targeting RNAi activators to liver cells. Mono- and tricistronic artificial primary microRNAs (pri-miRs) derived from pri-miR-31, placed under transcriptional control of the liver-specific modified murine transthyretin (mTTR) promoter, caused efficient inhibition of HBV replication markers. The tricistronic cassette was capable of silencing a mutant viral target and the effects were observed without disrupting the function of an endogenous miR (miR-16). The mTTR promoter stably expressed a reporter transgene in mouse livers over a study period of 1 year. Good silencing of HBV genes, without evidence of toxicity, was demonstrated following intravenous injection of LVs into neonatal HBV transgenic mice. Collectively, these data indicate that LVs may achieve sustained inhibition of HBV replication that is appealing for their therapeutic use.

Opportunities for treating chronic hepatitis B and C virus infection using RNA interference.

Activating the RNA interference (RNAi) pathway to achieve silencing of specific genes is one of the most exciting new developments of molecular biology. A particularly interesting use of this technology is inhibition of defined viral gene expression. In this review, we discuss the potential application of RNAi to treatment of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Globally, these hepatotropic viruses are the most important causes of cirrhosis and liver cancer. Available treatments have their limitations, which makes development of novel effective RNAi-based therapies for HBV and HCV especially significant. Several investigations carried out in vitro and in vivo are summarized, which demonstrate proof of principle that HBV and HCV can be inhibited by RNAi activators. Challenges facing further development of this technology to a stage of clinical application are discussed.

Downregulation of E-cadherin by hepatitis B virus X antigen in hepatocellullar carcinoma.

Hepatitis B virus (HBV)-encoded X antigen (HBxAg) contributes to the development of hepatocellular carcinoma (HCC). A frequent characteristic of HCC is reduced or absent expression of the cell adhesion protein, E-cadherin, although it is not known whether HBxAg plays a role. To address this, the levels of E-cadherin were determined in HBxAg-positive and -negative HepG2 cells in culture, and in tumor and surrounding nontumor liver from a panel of HBV carriers. The results showed an inverse relationship between HBxAg and E-cadherin expression both in tissue culture and in vivo. In HBxAg-positive cells, E-cadherin was suppressed at both the mRNA and protein levels. This was associated with hypermethylation of the E-cadherin promoter. Depressed E-cadherin correlated with HBxAg trans-activation function, as did the migration of HepG2 cells in vitro. Decreased expression of E-cadherin was also associated with the accumulation of beta-catenin in the cytoplasm and/or nuclei in tissues and cell lines, which is characteristic of activated beta-catenin. Additional work showed that HBxAg-activated beta-catenin. Together, these results suggest that the HBxAg is associated with decreased expression of E-cadherin, accumulation of beta-catenin in the cytoplasm and nucleus, and increased cell migration, which may contribute importantly to hepatocarcinogenesis.

Hepatitis B virus and hepatocellular carcinoma.

Chronic hepatitis B virus (HBV) infection is a major global cause of hepatocellular carcinoma (HCC). Individuals who are chronic carriers have a greater than 100-fold increased relative risk of developing the tumour. Several mechanisms of HBV-induced HCC have been proposed. Integration of HBV DNA into the genome of hepatocytes occurs commonly, although integration at cellular sites that are important for regulation of hepatocyte proliferation appears to be a rare event. Functions of the HBx protein are also potentially oncogenic. These include transcriptional activation of cellular growth regulatory genes, modulation of apoptosis and inhibition of nucleotide excision repair of damaged cellular DNA. The effects of HBx are mediated by interaction with cellular proteins and activation of cell signalling pathways. Variations in HBV genome sequences may be important in hepatocarcinogenesis, although their significance has not yet been completely elucidated. Necroinflammatory hepatic disease, which often accompanies chronic HBV infection, may contribute indirectly to hepatocyte transformation in a number of ways, including by facilitating HBV DNA integration, predisposing to the acquisition of cellular mutations and generating mutagenic oxygen reactive species. Although HCC is a malignancy with a poor prognosis, the availability of an effective vaccine against HBV infection, and its inclusion in the Expanded Programme of Immunization of many countries, augurs well for the eventual elimination of HBV-associated HCC.

A cellular gene up-regulated by hepatitis B virus-encoded X antigen promotes hepatocellular growth and survival.

Polymerase chain reaction (PCR) select complementary DNA (cDNA) subtraction of hepatitis B x antigen (HBxAg)-positive compared with -negative HepG2 cells resulted in the up-regulated expression of a cellular gene that encodes a transcript of 745 bases and a polypeptide 99 amino acids long. GenBank analysis revealed extensive homology with the amino terminal domain of cellular multidrug resistant proteins (MRP), although overexpression of this gene did not confer an MRP phenotype. In situ hybridization and immunostaining showed colocalized expression with HBxAg in the liver of hepatitis B carriers. Overexpression of this protein stimulated the growth of HepG2 cells in serum-free medium, and partially protected cells from anti-Fas-mediated killing, but did not promote growth in soft agar or tumor formation in nude mice. Introduction of the dominant negative inhibitor of nuclear factor kappaB (IkappaBalpha) into HBxAg-positive HepG2 cells decreased the levels of messenger RNA (mRNA) and protein, suggesting that its up-regulation is nuclear factor kappaB (NF-kappaB) dependent. Hence, HBxAg activation of NF-kappaB may result in the up-regulation of a cellular protein that promotes growth factor-independent survival and protects against Fas-mediated killing. This factor may contribute to the persistence of infected hepatocytes during chronic infection, which is important for the later development of hepatocellular carcinoma (HCC).

Hammerhead ribozyme-mediated inhibition of hepatitis B virus X gene expression in cultured cells.

BACKGROUND/AIMS: Chronic infection with hepatitis B virus (HBV) is endemic to sub-Saharan Africa and parts of Asia. Common complications of HBV persistence include cirrhosis and hepatocellular carcinoma (HCC). Present treatment of chronic HBV infection is usually ineffective and novel therapeutic approaches are an important objective. The HBV X protein (HBx) is a transcriptional activator that is required for the establishment of HBV infection and is implicated in hepatocarcinogenesis. The aim of this study was to assess the ability of two endogenously expressed hammerhead ribozymes to inhibit expression of HBV genes in transfected cultured cells. METHODS: Eukaryotic expression plasmids producing two ribozymes targeted to the HBx open reading frame, as well as their catalytically inactive homologues, were generated. Established cell lines and a primary culture of malignant hepatocytes were transfected to assess ribozyme effects on HBx expression and HBV replication. RESULTS: The ribozyme-expressing vectors inhibit expression of functional HBx protein and decrease HBV mRNA encoding surface and HBx sequences in transfected cells. Moreover, decreased HBsAg and HBeAg secretion from cells transfected with the ribozymes and an HBV replication competent plasmid provide evidence for an antireplicative effect of the ribozymes. However, the data do not exclude a dominant antisense effect that inhibits HBV gene expression. CONCLUSIONS: Inactivation of HBx, a sequence that is conserved in mammalian hepadnaviruses and found in all HBV transcripts, has potential for the treatment of chronic HBV infection.

Putative role of hepatitis B virus X protein in hepatocarcinogenesis: effects on apoptosis, DNA repair, mitogen-activated protein kinase and JAK/STAT pathways.

Chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The pathogenesis of HBV-induced malignant transformation is, however, incompletely understood. HBx, the protein encoded by the X open reading frame, is a transcriptional activator that has been implicated in hepatocarcinogenesis. HBx inhibits the function of the tumour suppressor protein p53 in what is thought to be an early event in hepatocyte transformation before the later accumulation of inactivating p53 point mutations. HBx inhibits apoptosis but also exerts pro-apoptotic effects. The effects of HBx on apoptosis may be important not only for the development of HCC but also for the establishment of HBV infection. Further implication of HBx in hepatocyte transformation has been the demonstration that it inhibits the repair of damaged hepatocyte DNA. This effect may be mediated by interaction with p53 or through binding to the damaged DNA binding protein (DDB), which plays an accessory role in nucleotide excision repair. In addition, HBx activates cell signalling cascades involving mitogen-activated protein kinase (MAPK) and Janus family tyrosine kinases (JAK)/signal transducer and activators of transcription (STAT) pathways. The implications of these modulating effects of HBx are not fully understood, but they are likely to have wide-ranging effects on hepatocyte proliferation, apoptosis and the regulation of cell growth checkpoints. The cellular functions ascribed to HBx are unusually diverse, and defining the biologically important role of HBx during HBV replication will go some way to understanding the sequelae of chronic HBV infection.

In situ demonstration of inhibitory effects of hammerhead ribozymes that are targeted to the hepatitis Bx sequence in cultured cells.

Chronic hepatitis B virus (HBV) infection is endemic to several populous areas of the world and is frequently complicated by hepatocellular carcinoma. Ribozymes can be designed to cleave target RNA sequences specifically and show promise for the treatment of HBV infection. Demonstration of intracellular inhibition of HBV gene expression, essential to developing therapeutic ribozymes, has been the aim of this investigation. We generated two vectors encoding hammerhead ribozymes that target the HBx region of HBV. Plasmids containing intact HBV sequences or a modification in which the preS2/S region was replaced by DNA encoding enhanced green fluorescent protein (EGFP) were used to test ribozyme action in transfected cells. Both ribozymes inhibited surface antigen secretion and EGFP expression similarly. The measurement of EGFP expression is convenient to assess ribozyme action in situ and effective targeting of HBV sequences that are common to all HBV transcripts is potentially useful to develop strategies to counter HBV infection.

The translation initiation factor, hu-Sui1 may be a target of hepatitis B X antigen in hepatocarcinogenesis.

The role of hepatitis B virus X antigen in the development of hepatocellular carcinoma was explored by stably transfecting HepG2 cells with an X antigen expression vector, and identifying the differences in gene expression that distinguish X positive from X negative cells by subtractive PCR. One differentially expressed gene, the human homolog of sui1 (hu-sui1), encodes a translation initiation factor whose expression was suppressed by X antigen in HepG2 cells. Hu-Sui1 was also expressed in nontumor liver but not in tumor cells from patients with hepatocellular carcinoma. Introduction of hu-sui1 into HepG2 cells inhibited cell growth in culture, in soft agar, and partially inhibited tumor formation in nude mice. Hence, the suppression of hu-sui1 by X antigen may result in the abrogation of negative growth regulation and contribute to the development of hepatocellular carcinoma.

Hepatitis B virus X-protein binds damaged DNA and sensitizes liver cells to ultraviolet irradiation.

The mechanism which is responsible for the association of chronic hepatitis B virus (HBV) infection with hepatocellular carcinoma (HCC) is poorly understood. The protein encoded by the HBV X-gene (HBx) has been identified as potentially oncogenic. HBx is a promiscuous indirect trans-activator of a wide range of cellular and viral cis-elements and may disrupt the maintenance of genomic integrity by inhibiting p53 function and binding a putative DNA repair protein (XAP-1). In this report, we show that there is preferential binding of recombinant HBx to damaged DNA through an association with nuclear proteins. We have used the transcriptional activation by HBx of the beta-actin promoter of a beta-galactosidase reporter cassette to label cultured Chang liver cells expressing HBx. We demonstrate that cells expressing HBx are sensitised to the lethal effects of low dose ultraviolet irradiation. These data indicate that HBx interferes with liver cell DNA repair by binding damaged DNA and may predispose to the accumulation of potentially lethal or carcinogenic mutations.

In vitro and in vivo hepatoma cell-specific expression of a gene transferred with an adenoviral vector.

Recombinant adenoviruses are widely used for the transfer of foreign genes into various mammalian cells. However, the utilization of these vectors for cancer gene therapy requires the specific and efficient expression of the transferred gene in tumor cells. To obtain targeted expression in hepatoma cells, we constructed recombinant adenoviral vectors containing transcriptional elements from either the rat alpha-fetoprotein (AFP) or the human insulin-like growth factor II (IGFII) genes driving expression of the nuclear beta-galactosidase gene (nls lacZ). In vitro infection revealed that the AFP but not the IGFII transcriptional regulatory sequence controlled nls lacZ expression specifically in hepatoma cells. The same specificity was obtained in vivo in subcutaneous human hepatic tumors generated by engraftment of Huh7 hepatoma cells in nude mice as well as in primary liver tumors developed in rats and mice. No marker gene expression was detectable after AFP-nls lacZ gene transfer to normal rat liver parenchyma despite evidence for the presence of DNA encoding the nls lacZ gene. However, in vivo experiments with primary liver tumors in rats and mice also revealed that primary hepatoma cells were poorly infected by adenoviral vectors. Peritumoral and normal tissues were infected efficiently by adenoviral vectors. We conclude that hepatoma cell-specific expression of a transgene can be achieved with AFP regulatory sequences but that adenoviral vectors may not be the preferable vector for transferring genes in vivo in primary liver tumors.

Hepatoma cell-specific expression of a retrovirally transferred gene is achieved by alpha-fetoprotein but not insulinlike growth factor II regulatory sequences.

To target gene expression to malignant hepatic cells, we have constructed recombinant retroviral vectors containing a reporter gene encoding nuclear beta-galactosidase (nls-LacZ) under transcriptional control of regulatory sequences from the rat alpha-fetoprotein (AFP) or human insulinlike growth factor II (IGFII) genes. The AFP and IGFII P3 promoters activate transcription during fetal development and are often reactivated in hepatocellular carcinoma (HCC). Infection of several cultured cell types with the retroviral vector containing the IGFII P3 sequence resulted in expression of the reporter gene in all cell lines tested, including those that do not produce IGFII. In contrast, selective expression was achieved by vectors containing the AFP transcriptional regulatory sequence. Nuclear beta-galactosidase activity was detectable in cells from lines that produce AFP, and not in cells that do not express the AFP gene. In most infected cell lines, retroviral RNA synthesis from the 5' LTR was inhibited, and deletion of the retroviral LTR enhancer did not change expression from either the IGFII P3-nls-LacZ or the AFP-nls-LacZ cassettes. After treatment of cells with 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor (EGF), the decrease in concentrations of endogenous AFP messenger RNA (mRNA) and nls-LacZ mRNA transcribed from the transferred AFP regulatory sequence were similar. In the context of an integrated provirus, the AFP transcriptional regulatory sequence is therefore subject to similar regulatory control as that of the endogenous gene. These data show that the AFP sequence, and not the IGFII P3 promoter we used, is suitable for targeting gene expression to malignant hepatic cells.