Macrophage derived TNFα promotes hepatic reprogramming to Warburg-like metabolism.

During infection, hepatocytes must undergo a reprioritization of metabolism, termed metabolic reprogramming. Hepatic metabolic reprogramming in response to infection begins within hours of infection, suggesting a mechanism closely linked to pathogen recognition. Following injection with polyinosinic:polycytidylic acid, a mimic of viral infection, a robust hepatic innate immune response could be seen involving the TNFα pathway at 2 h. Repeated doses led to the adoption of Warburg-like metabolism in the liver as determined by in vivo metabolic imaging, expression analyses, and metabolomics. Hepatic macrophages, Kupffer cells, were able to induce Warburg-like metabolism in hepatocytes in vitro via TNFα. Eliminating macrophages in vivo or blocking TNFα in vitro or in vivo resulted in abrogation of the metabolic phenotype, establishing an immune-metabolic axis in hepatic metabolic reprogramming. Overall, we suggest that macrophages, as early sensors of pathogens, instruct hepatocytes via TNFα to undergo metabolic reprogramming to cope with challenges to homeostasis initiated by infection. This work not only addresses a key component of end-organ physiology, but also raises questions about the side effects of biologics in the treatment of inflammatory diseases. KEY MESSAGES: • Hepatocytes develop Warburg-like metabolism in vivo during viral infection. • Macrophage TNFα promotes expression of glycolytic enzymes in hepatocytes. • Blocking this immune-metabolic axis abrogates Warburg-like metabolism in the liver. • Implications for patients being treated for inflammatory diseases with biologics.

Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy.

The use of adeno-associated virus (AAV) as a gene therapy vector has been approved recently for clinical use and has demonstrated efficacy in a growing number of clinical trials. However, the safety of AAV as a vector has been challenged by a single study that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. Most studies have not noted genotoxicity following AAV-mediated gene delivery; therefore, the possibility that there is an association between AAV and HCC is controversial. Here, we performed a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the RNA imprinted and accumulated in nucleus (Rian) locus, and the resulting overexpression of proximal microRNAs and retrotransposon-like 1 (Rtl1) were associated with HCC. In addition, we demonstrated that the AAV vector dose, enhancer/promoter selection, and the timing of gene delivery are all critical factors for determining HCC incidence after AAV gene delivery. Together, our results define aspects of AAV-mediated gene therapy that influence genotoxicity and suggest that these features should be considered for design of both safer AAV vectors and gene therapy studies.

Species D adenoviruses as oncolytics against B-cell cancers.

PURPOSE: Oncolytic viruses are self-amplifying anticancer agents that make use of the natural ability of viruses to kill cells. Adenovirus serotype 5 (Ad5) has been extensively tested against solid cancers, but less so against B-cell cancers because these cells do not generally express the coxsackie and adenoviral receptor (CAR). To determine whether other adenoviruses might have better potency, we "mined" the adenovirus virome of 55 serotypes for viruses that could kill B-cell cancers. EXPERIMENTAL DESIGN: Fifteen adenoviruses selected to represent Ad species B, C, D, E, and F were tested in vitro against cell lines and primary patient B-cell cancers for their ability to infect, replicate in, and kill these cells. Select viruses were also tested against B-cell cancer xenografts in immunodeficient mice. RESULTS: Species D adenoviruses mediated most robust killing against a range of B-cell cancer cell lines, against primary patient marginal zone lymphoma cells, and against primary patient CD138+ myeloma cells in vitro. When injected into xenografts in vivo, single treatment with select species D viruses Ad26 and Ad45 delayed lymphoma growth. CONCLUSIONS: Relatively unstudied species D adenoviruses have a unique ability to infect and replicate in B-cell cancers as compared with other adenovirus species. These data suggest these viruses have unique biology in B cells and support translation of novel species D adenoviruses as oncolytics against B-cell cancers.

Adeno-associated virus serotype 8 gene transfer rescues a neonatal lethal murine model of propionic acidemia.

Propionic acidemia (PA) is an autosomal recessive disorder of metabolism caused by a deficiency of propionyl-coenzyme A carboxylase (PCC). Despite optimal dietary and cofactor therapy, PA patients still suffer from lethal metabolic instability and experience multisystemic complications. A murine model of PA (Pcca(-/-)) of animals that uniformly die within the first 48 hr of life was used to determine the efficacy of adeno-associated viral (AAV) gene transfer as a potential therapy for PA. An AAV serotype 8 (AAV8) vector was engineered to express the human PCCA cDNA and delivered to newborn mice via an intrahepatic injection. Greater than 64% of the Pcca(-/-) mice were rescued after AAV8-mediated gene transfer and survived until day of life 16 or beyond. Western analysis of liver extracts showed that PCC was completely absent from Pcca(-/-) mice but was restored to greater than wild-type levels after AAV gene therapy. The treated Pcca(-/-) mice also exhibited markedly reduced plasma levels of 2-methylcitrate compared with the untreated Pcca(-/-) mice, which indicates significant PCC enzymatic activity was provided by gene transfer. At the time of this report, the oldest treated Pcca(-/-) mice are over 6 months of age. In summary, AAV gene delivery of PCCA effectively rescues Pcca(-/-) mice from neonatal lethality and substantially ameliorates metabolic markers of the disease. These experiments demonstrate a gene transfer approach using AAV8 that might be used as a treatment for PA, a devastating and often lethal disorder desperately in need of new therapeutic options.

Infection and killing of multiple myeloma by adenoviruses.

Oncolytic virotherapy makes use of the natural ability of viruses to infect and kill cancer cells. Adenovirus serotype 5 (Ad5) has been approved for use in humans as a therapy for solid cancers. In this study, we have tested whether Ad5 and low-seroprevalence adenoviruses can be used as oncolytics for multiple myeloma (MM). We show that Ad5 productively infects most myeloma cell lines, replicates to various degrees, and mediates oncolytic cell killing in vitro and in vivo. Comparison of Ad5 with low-seroprevalence Ads on primary marrow samples from MM patients revealed striking differences in the abilities of different adenoviral serotypes to kill normal CD138(-) cells and CD138(+) MM cells. Ad5 and Ad6 from species C and Ad26 and Ad48 from species D all mediated killing of CD138(+) cells with low-level killing of CD138(-) cells. In contrast, Ad11, Ad35, Ad40, and Ad41 mediated weak oncolytic effects in all of the cells. Comparison of cell binding, cell entry, and replication revealed that Ad11 and Ad35 bound MM cells 10 to 100 times better than other serotypes. However, after this efficient interaction, Ad11 and Ad35 viral DNA was not replicated and cell killing did not occur. In contrast, Ad5, Ad6, Ad26, and Ad48 all replicated 10- to 100-fold in MM cells and this correlated with cell killing. These data suggest that Ad5 and other low-seroprevalence adenoviruses may have utility as oncolytic agents against MM and other hematologic malignancies.

Systemic delivery of therapeutic viruses.

The treatment of certain diseases will require the systemic delivery of therapeutic genes or viruses. In most cases, intravascular injection is the best delivery method to achieve the systemic distribution of viruses and to enable these agents to reach distant therapeutic sites. However, viruses administered by intravascular injection encounter overlapping barriers that impede their ability to reach their targets, including interactions with blood cells, blood factors and endothelial cells, loss to hepatocytes and macrophages, and destruction by innate and adaptive immune responses. In this review, recent advances in the understanding of the mechanisms determining virus tropism following systemic administration and the pharmacology of therapeutic viruses are described. Adenoviruses are used as a paradigm of these interactions, and factors affecting their therapeutic efficacy and side effects are discussed, as well as how the barriers that impede their ability to reach their targets translate to other therapeutic viruses.

Comparison of replication-competent, first generation, and helper-dependent adenoviral vaccines.

All studies using human serotype 5 Adenovirus (Ad) vectors must address two major obstacles: safety and the presence of pre-existing neutralizing antibodies. Helper-Dependent (HD) Ads have been proposed as alternative vectors for gene therapy and vaccine development because they have an improved safety profile. To evaluate the potential of HD-Ad vaccines, we compared replication-competent (RC), first-generation (FG) and HD vectors for their ability to induce immune responses in mice. We show that RC-Ad5 and HD-Ad5 vectors generate stronger immune responses than FG-Ad5 vectors. HD-Ad5 vectors gave lower side effects than RC or FG-Ad, producing lower levels of tissue damage and anti-Ad T cell responses. Also, HD vectors have the benefit of being packaged by all subgroup C serotype helper viruses. We found that HD serotypes 1, 2, 5, and 6 induce anti-HIV responses equivalently. By using these HD serotypes in heterologous succession we showed that HD vectors can be used to significantly boost anti-HIV immune responses in mice and in FG-Ad5-immune macaques. Since HD vectors have been show to have an increased safety profile, do not possess any Ad genes, can be packaged by multiple serotype helper viruses, and elicit strong anti-HIV immune responses, they warrant further investigation as alternatives to FG vectors as gene-based vaccines.

Short-term rescue of neonatal lethality in a mouse model of propionic acidemia by gene therapy.

Propionic acidemia (PA) is a metabolic disorder that causes mental retardation and that can be fatal if untreated. PA is inherited in an autosomal recessive fashion involving mutations in PCCA or PCCB encoding the alpha and beta subunits of propionyl-CoA carboxylase (PCC). Current treatment is based on dietary restriction of substrate amino acids, which attenuates symptoms. However, patients still experience episodes of hyperammonemia that can cause progressive neurologic damage. In this paper, we have tested gene therapy approaches to PA in a stringent mouse model of PCCA deficiency, in which homozygous knockout mice are born but die within 36 hr. In this work, we have delivered first-generation and helper-dependent adenovirus serotype 5 (Ad5) vectors expressing the human PCCA cDNA by intraperitoneal injection into newborn mice. Unmodified Ad5 vectors mediated extensive transduction of the peritoneum with weak liver transduction as determined by luciferase imaging and dsRed expression. In contrast, modification of Ad5 with polyethylene glycol detargeted the virus from the peritoneum and retargeted it for transduction in the liver. When vectors expressing PCCA were injected, significant increases in life span were observed for both the unmodified and polyethylene glycol (PEG)-modified Ad5 vectors. However, this rescue was transient. Similarly, adeno-associated virus serotype 8-mediated transduction also produced only transient rescue. These data show first proof of principle for gene therapy of PA and demonstrate the potential utility of PEG to modify viral tropism in an actual gene therapy application.

Macrophage depletion combined with anticoagulant therapy increases therapeutic window of systemic treatment with oncolytic adenovirus.

Liver tropism of systemically delivered adenoviruses (Ad) represents a considerable challenge for their use as anticancer therapeutics. More than 90% of i.v. injected Ad is rapidly taken up by the liver leading to hepatotoxicity, reduced virus uptake by target tumor tissue, and diminished therapeutic efficacy. The lack of clinical activity of systemically given oncolytic Ad demands for better understanding and improvement of virus pharmacokinetics. We studied the effects of Ad "detargeting" from liver macrophages (Kupffer cells) and hepatocytes on toxicity and anticancer efficacy using a nonattenuated oncolytic Ad expressing enhanced green fluorescent protein-firefly luciferase fusion protein (Ad-EGFPLuc). Kupffer cell depletion before i.v. injection of Ad-EGFPLuc increased transgene expression in the liver 40.7-fold on day 3 after the injection indicating compensatory enhancement of hepatocyte transduction due to increased bioavailability of the virus. Pretreatment of mice with the anticoagulant drug warfarin to block blood factor-dependent binding of the virus to hepatocytes markedly reduced luciferase expression in the liver and mediated the corresponding decrease of hepatotoxicity in mice with intact and depleted liver macrophages. Combined depletion of Kupffer cells and pretreatment with warfarin before a single i.v. injection of Ad-EGFPLuc significantly reduced tumor growth and prolonged survival of nude mice bearing subcutaneous xenografts of aggressive human hepatocellular carcinoma. The improved antitumor activity correlated with enhanced transgene expression and virus spread in the tumors. These data suggest that detargeting oncolytic Ad from liver macrophages and hepatocytes is an effective strategy to increase the therapeutic window for therapy against disseminated tumor sites.