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Systemic levels of liver enzymes as indicators of liver damage. Systemic levels of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) liver enzyme levels were measured from plasma for all experimental groups of C57BL/6 mice 10 days after Ad- LacZ or Ad-GFP delivery (i.e. during the acute inflammatory response in the liver) and compared to data from AAV-LacZ transduced mice that did not receive Ad vectors. Data are average 6 SD for n = 4 mice/ experimental group. doi:10.1371/journal.pone.0006376.g004
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Hepatic gene transfer, in particular using adeno-associated viral (AAV) vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppr...
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... to determine level of ALT (alanine aminotransferase) and AST (aspartate aminotransferase), which are indicators of liver damage. ALT and AST levels were measured by the clinical chemistry lab at the small animal hospital affiliated with the University of Florida Veterinary Program (Gainesville, FL). Blood was collected from AAV-LacZ only, Ad-LacZ only, and AAV-LacZ plus Ad-LacZ transduced mice at Day 0, Day 14, Day 28 and Day 45 after IV delivery of Ad-LacZ. IgG1 and IgG2a immunocapture assays to determine antibody titers against b -gal was performed as follows. ELISA plates were coated with 1 ng/ m l of recombinant b -gal protein (Sigma, St Louis, MO) overnight at 4 u C. In parallel, 2-fold serial dilutions of mouse IgG1 or IgG2a (Sigma) were used to coat wells for standard curve. Blocking was done with dilution buffer (PBS containing 5% BSA and 0.05% Tween20) for 1 hr at room temperature. Samples were added at 1:20 for 2 hours at 37 u C. Goat anti-mouse IgG1 or IgG2a HRP- conjugated secondary antibody (Southern Biotech, Birmingham, AL) was added at 1:2000 in dilution buffer for 2 hours at 37 u C. Detection was done using SIGMA FAST TM OPD tablets (Sigma). Absorption (OD 450 ) was measured using the Model 680 micro- plate reader (Bio-Rad, Hercules, CA). Statistical comparisons between experimental groups were performed by two-tailed Student’s t test. Values were considered to be statistically significant for P , 0.05. In previous studies, we have demonstrated induction of immune tolerance to coagulation factor IX (F.IX) by hepatic AAV gene transfer [22]. In this study, we chose b -gal as a model antigen for a cytoplasmic expressed protein. Gene transfer was performed in C57BL/6 mice for the following reasons. This strain mounts effective CD8 + T cell responses against b -gal expressing cells (such as hepatocytes or myofibers) upon adenoviral gene transfer; it is known that following AAV gene transfer to skeletal muscle, lacZ expression in myofibers of C57BL/6 mice persists because of ignorance but is eliminated by a CTL response upon secondary + gene transfer with Ad-LacZ; the immunodominant CD8 T cell epitope for b -gal is known [3,4,31–34]. An outline of our experimental approach is described in Fig. 1. First, the capacity for hepatic AAV gene transfer to develop tolerance to a cytoplasmic transgene product and to protect against immunotoxic responses was explored. AAV-LacZ transduced livers showed a low level of transgene expression in 1–3% of hepatocytes when analyzed 45 days after gene transfer (Fig. 2). Ad- LacZ gene transfer resulted in transient high-level b -gal activity (40–75% of hepatocytes) at 10 days, which, as expected, declined to undetectable by 45 days (Fig. 2A-C). In contrast, livers initially transduced with AAV-LacZ had b -gal expression in 35–78% of hepatocytes and continued to express in a range of 4%–14% when analyzed 45 days after secondary gene transfer with Ad-LacZ (which was performed 45 days after AAV-LacZ transduction). These results demonstrate that a portion of the additional b -gal expression introduced by the more effective but highly immunogenic Ad-LacZ vector remained protected. This is in contrast to findings by others on muscle gene transfer, showing that secondary Ad-LacZ gene transfer eliminates previously AAV-LacZ transduced skeletal muscle fibers [4]. Since we observed partial protection of Ad-LacZ transduced hepatocytes, we decided to assess the level of hepatotoxicity induced by Ad-LacZ in na ̈ve control and AAV-LacZ pre-treated mice (n = 4 per group) by assessment of liver inflammation and measuring the systemic levels of liver enzymes as indicators of liver damage. In mice that had received AAV-LacZ only, there were 2 cases of mild inflammation and 2 cases of moderate inflammation in the parenchyma, and 4/4 animals had only mild inflammation in the portal ducts (Fig. 3A, B, I, J). All (4/4) Ad- LacZ only transduced livers presented with severe inflammation in the parenchyma and 2 cases of moderate and 2 cases of severe inflammation in the portal ducts (Fig. 3G, H, I, J). Additional control groups transduced with Ad-GFP vector only or receiving AAV-LacZ followed by Ad-GFP showed similar levels of severe inflammation (Fig. 3E, F, I, J). The AAV-LacZ/Ad-LacZ group, however, showed 4 of 4 livers with moderate inflammation in the parenchyma and 3 cases of moderate and 1 case of mild inflammation in the portal ducts (Fig. 3 C, D, I, J). In summary, these results reflected a level of inflammation that was intermediate between AAV-LacZ only and Ad-LacZ only treated mice. Systemic ALT and AST levels, indicators of liver damage, were within normal range (40 U/L and 70 U/L, respectively) in the AAV-LacZ only group (Fig. 4). In the AAV2-LacZ/Ad-LacZ group, ALT levels were elevated to 250 u/L and AST levels elevated to 225 U/L, but were significantly reduced compared to the AAV-LacZ/Ad-GFP, Ad-LacZ only, and Ad-GFP only groups, which were 350 u/L (ALT) and 325–375 U/L (Fig. 4). These measurements were performed on plasma samples obtained 10 days after adenoviral gene transfer. It is known that administration of a first generation adenoviral + vector causes CD8 T cell responses against transgene product and viral antigens encoded by the vector backbone. Co-staining of CD8 + cells and transgene expressing cells (either b -gal or GFP) + was used to determine levels of CD8 cellular infiltrate at the sight of transgene production at 10 days after Ad-LacZ or Ad-GFP vector delivery. Immunofluorescent detection showed close to a 2:1 ratio of CD8 + cells to either b -gal + or GFP + hepatocytes in livers of Ad-LacZ, Ad-GFP, or AAV-LacZ/Ad-GFP transduced mice, while this ratio was 0.2:1 in the AAV-LacZ/Ad-LacZ group. Therefore, this difference represents a 10-fold decrease in the + CD8 cellular infiltrate in the AAV-LacZ/Ad-LacZ mice (Fig. 5). Taken together, the results shown in Figs. 2–4 demonstrate that tolerance induction to b -gal by AAV gene transfer substantially reduces transgene product-directed immunotoxicity and that the reduction of adenoviral gene transfer-induced liver toxicity and hepatic CD8 + T cell infiltrate is linked to b -gal transgene expression. Toxicity and T cell responses to the liver remain high in secondary gene transfer with an Ad vector expressing a different transgene product. Ad-LacZ transduced mice on average produced 500–700 ng/ ml plasma of IgG1 anti- b -gal at days 14, 28, and 45 after gene transfer (Fig. 6). No IgG2a anti- b -gal was detected (data not shown; this is different from muscle-directed Ad-LacZ gene transfer, which results in IgG2a formation) [19]. In contrast, both AAV-LacZ only and AAV-LacZ/Ad-LacZ transduced mice failed to form antibodies against b -gal (Fig. 6). In order to quantify the effect of tolerance induction on activation of pro-inflammatory subsets of T cells, ELISpot assays were performed 14 days after Ad-LacZ gene transfer to na ̈ve or AAV-LacZ pre-treated mice. Splenocytes from individual mice were re-stimulated in vitro with b -gal, the immunodominant b -gal + CD8 T cell epitopes, or heat-inactivated adenoviral particles. Frequencies of IFN- c secreting cells were measured for Ad-LacZ mice only and AAV-LacZ/Ad-LacZ treated mice (Fig. 7A). AAV- LacZ only mice served as an additional control. Ad-LacZ induced a robust CD8 + T cell response to b -gal (9-fold above the frequency in mock-stimulated cultures), which was nearly undetectable in AAV-LacZ or AAV-LacZ/Ad-LacZ transduced animals. Similar results were obtained for the IFN- c + response to entire b -gal protein antigen (Fig. 7A). Ad-lacZ only and AAV-LacZ/Ad-LacZ transduced mice showed an IFN- c + response to adenoviral particles, which was somewhat reduced in the AAV-LacZ/Ad- LacZ group (Fig. 7A). All splenocyte cultures showed equally high responses to control stimulation with super antigen (Fig. 7B). Adoptively transferred CD4 CD25 splenocytes from AAV- LacZ and AAV-LacZ/Ad-LacZ but not Ad-LacZ transduced mice were able to significantly suppress the IFN- c + response against the dominant b -gal CD8 + T cell epitope when compared to non- specific Treg transferred from na ̈ve mice (Fig. 7C). In contrast, IFN- c + responses to control stimulation with super antigen were not suppressed (Fig. 7D). + + Furthermore, we found that the frequency of CD25 Foxp3 + Treg among CD4 T cells was significantly increased in AAV- LacZ transduced mice following secondary Ad-LacZ gene transfer (Fig. 8A,B), which was not seen for secondary gene transfer with Ad-GFP control vector. Ten days after adenoviral gene transfer, the percentages of Treg were lowest in spleens of those mice that had been transduced with adenoviral vectors only, irrespective of the transgene. Finally, we wanted to test for induction of a Treg response in the liver, which is technically more challenging. Therefore, utilized FoxP3 reporter mice (FoxP3-IRES-EGFP knock-in mice) to + analyze for frequencies of Treg in form of EGFP intrahepatic lymphocytes 10 days after Ad-LacZ gene transfer. In AAV-LacZ pre-treated mice, the frequencies of Treg were significantly increased compared to AAV-LacZ transduced mice that did not receive the secondary gene transfer (Fig. 8C), while Treg were undetectable among hepatic lymphocytes from Ad-LacZ only treated mice. Hepatic gene transfer with AAV vectors has been shown to induce tolerance to a number of transgene products in animal models of human disease. This treatment provided immune tolerance to the therapeutic protein and, simultaneously, therapy or, alternatively, allowed for safe administration of supplementary therapy such as enzyme replacement therapy or therapeutic gene transfer to a different organ [35–37]. Using this method, animals have been tolerized to F.IX, a -galacosidase, acid a -glucosidase, acid shingomyelinase, and other proteins [38]. Interestingly, these are secreted or exocytosed proteins that have been expressed in hepatocytes primarily for the ...
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... to determine the level of CD8 + and b -gal + staining. The ratio of hepatocytes producing + the transgene product and CD8 cells was determined using Pro Image Software. Blood was collected and plasma isolated from mice at day 10 after Ad I.V. gene transfer to determine level of ALT (alanine aminotransferase) and AST (aspartate aminotransferase), which are indicators of liver damage. ALT and AST levels were measured by the clinical chemistry lab at the small animal hospital affiliated with the University of Florida Veterinary Program (Gainesville, FL). Blood was collected from AAV-LacZ only, Ad-LacZ only, and AAV-LacZ plus Ad-LacZ transduced mice at Day 0, Day 14, Day 28 and Day 45 after IV delivery of Ad-LacZ. IgG1 and IgG2a immunocapture assays to determine antibody titers against b -gal was performed as follows. ELISA plates were coated with 1 ng/ m l of recombinant b -gal protein (Sigma, St Louis, MO) overnight at 4 u C. In parallel, 2-fold serial dilutions of mouse IgG1 or IgG2a (Sigma) were used to coat wells for standard curve. Blocking was done with dilution buffer (PBS containing 5% BSA and 0.05% Tween20) for 1 hr at room temperature. Samples were added at 1:20 for 2 hours at 37 u C. Goat anti-mouse IgG1 or IgG2a HRP- conjugated secondary antibody (Southern Biotech, Birmingham, AL) was added at 1:2000 in dilution buffer for 2 hours at 37 u C. Detection was done using SIGMA FAST TM OPD tablets (Sigma). Absorption (OD 450 ) was measured using the Model 680 micro- plate reader (Bio-Rad, Hercules, CA). Statistical comparisons between experimental groups were performed by two-tailed Student’s t test. Values were considered to be statistically significant for P , 0.05. In previous studies, we have demonstrated induction of immune tolerance to coagulation factor IX (F.IX) by hepatic AAV gene transfer [22]. In this study, we chose b -gal as a model antigen for a cytoplasmic expressed protein. Gene transfer was performed in C57BL/6 mice for the following reasons. This strain mounts effective CD8 + T cell responses against b -gal expressing cells (such as hepatocytes or myofibers) upon adenoviral gene transfer; it is known that following AAV gene transfer to skeletal muscle, lacZ expression in myofibers of C57BL/6 mice persists because of ignorance but is eliminated by a CTL response upon secondary + gene transfer with Ad-LacZ; the immunodominant CD8 T cell epitope for b -gal is known [3,4,31–34]. An outline of our experimental approach is described in Fig. 1. First, the capacity for hepatic AAV gene transfer to develop tolerance to a cytoplasmic transgene product and to protect against immunotoxic responses was explored. AAV-LacZ transduced livers showed a low level of transgene expression in 1–3% of hepatocytes when analyzed 45 days after gene transfer (Fig. 2). Ad- LacZ gene transfer resulted in transient high-level b -gal activity (40–75% of hepatocytes) at 10 days, which, as expected, declined to undetectable by 45 days (Fig. 2A-C). In contrast, livers initially transduced with AAV-LacZ had b -gal expression in 35–78% of hepatocytes and continued to express in a range of 4%–14% when analyzed 45 days after secondary gene transfer with Ad-LacZ (which was performed 45 days after AAV-LacZ transduction). These results demonstrate that a portion of the additional b -gal expression introduced by the more effective but highly immunogenic Ad-LacZ vector remained protected. This is in contrast to findings by others on muscle gene transfer, showing that secondary Ad-LacZ gene transfer eliminates previously AAV-LacZ transduced skeletal muscle fibers [4]. Since we observed partial protection of Ad-LacZ transduced hepatocytes, we decided to assess the level of hepatotoxicity induced by Ad-LacZ in na ̈ve control and AAV-LacZ pre-treated mice (n = 4 per group) by assessment of liver inflammation and measuring the systemic levels of liver enzymes as indicators of liver damage. In mice that had received AAV-LacZ only, there were 2 cases of mild inflammation and 2 cases of moderate inflammation in the parenchyma, and 4/4 animals had only mild inflammation in the portal ducts (Fig. 3A, B, I, J). All (4/4) Ad- LacZ only transduced livers presented with severe inflammation in the parenchyma and 2 cases of moderate and 2 cases of severe inflammation in the portal ducts (Fig. 3G, H, I, J). Additional control groups transduced with Ad-GFP vector only or receiving AAV-LacZ followed by Ad-GFP showed similar levels of severe inflammation (Fig. 3E, F, I, J). The AAV-LacZ/Ad-LacZ group, however, showed 4 of 4 livers with moderate inflammation in the parenchyma and 3 cases of moderate and 1 case of mild inflammation in the portal ducts (Fig. 3 C, D, I, J). In summary, these results reflected a level of inflammation that was intermediate between AAV-LacZ only and Ad-LacZ only treated mice. Systemic ALT and AST levels, indicators of liver damage, were within normal range (40 U/L and 70 U/L, respectively) in the AAV-LacZ only group (Fig. 4). In the AAV2-LacZ/Ad-LacZ group, ALT levels were elevated to 250 u/L and AST levels elevated to 225 U/L, but were significantly reduced compared to the AAV-LacZ/Ad-GFP, Ad-LacZ only, and Ad-GFP only groups, which were 350 u/L (ALT) and 325–375 U/L (Fig. 4). These measurements were performed on plasma samples obtained 10 days after adenoviral gene transfer. It is known that administration of a first generation adenoviral + vector causes CD8 T cell responses against transgene product and viral antigens encoded by the vector backbone. Co-staining of CD8 + cells and transgene expressing cells (either b -gal or GFP) + was used to determine levels of CD8 cellular infiltrate at the sight of transgene production at 10 days after Ad-LacZ or Ad-GFP vector delivery. Immunofluorescent detection showed close to a 2:1 ratio of CD8 + cells to either b -gal + or GFP + hepatocytes in livers of Ad-LacZ, Ad-GFP, or AAV-LacZ/Ad-GFP transduced mice, while this ratio was 0.2:1 in the AAV-LacZ/Ad-LacZ group. Therefore, this difference represents a 10-fold decrease in the + CD8 cellular infiltrate in the AAV-LacZ/Ad-LacZ mice (Fig. 5). Taken together, the results shown in Figs. 2–4 demonstrate that tolerance induction to b -gal by AAV gene transfer substantially reduces transgene product-directed immunotoxicity and that the reduction of adenoviral gene transfer-induced liver toxicity and hepatic CD8 + T cell infiltrate is linked to b -gal transgene expression. Toxicity and T cell responses to the liver remain high in secondary gene transfer with an Ad vector expressing a different transgene product. Ad-LacZ transduced mice on average produced 500–700 ng/ ml plasma of IgG1 anti- b -gal at days 14, 28, and 45 after gene transfer (Fig. 6). No IgG2a anti- b -gal was detected (data not shown; this is different from muscle-directed Ad-LacZ gene transfer, which results in IgG2a formation) [19]. In contrast, both AAV-LacZ only and AAV-LacZ/Ad-LacZ transduced mice failed to form antibodies against b -gal (Fig. 6). In order to quantify the effect of tolerance induction on activation of pro-inflammatory subsets of T cells, ELISpot assays were performed 14 days after Ad-LacZ gene transfer to na ̈ve or AAV-LacZ pre-treated mice. Splenocytes from individual mice were re-stimulated in vitro with b -gal, the immunodominant b -gal + CD8 T cell epitopes, or heat-inactivated adenoviral particles. Frequencies of IFN- c secreting cells were measured for Ad-LacZ mice only and AAV-LacZ/Ad-LacZ treated mice (Fig. 7A). AAV- LacZ only mice served as an additional control. Ad-LacZ induced a robust CD8 + T cell response to b -gal (9-fold above the frequency in mock-stimulated cultures), which was nearly undetectable in AAV-LacZ or AAV-LacZ/Ad-LacZ transduced animals. Similar results were obtained for the IFN- c + response to entire b -gal protein antigen (Fig. 7A). Ad-lacZ only and AAV-LacZ/Ad-LacZ transduced mice showed an IFN- c + response to adenoviral particles, which was somewhat reduced in the AAV-LacZ/Ad- LacZ group (Fig. 7A). All splenocyte cultures showed equally high responses to control stimulation with super antigen (Fig. 7B). Adoptively transferred CD4 CD25 splenocytes from AAV- LacZ and AAV-LacZ/Ad-LacZ but not Ad-LacZ transduced mice were able to significantly suppress the IFN- c + response against the dominant b -gal CD8 + T cell epitope when compared to non- specific Treg transferred from na ̈ve mice (Fig. 7C). In contrast, IFN- c + responses to control stimulation with super antigen were not suppressed (Fig. 7D). + + Furthermore, we found that the frequency of CD25 Foxp3 + Treg among CD4 T cells was significantly increased in AAV- LacZ transduced mice following secondary Ad-LacZ gene transfer (Fig. 8A,B), which was not seen for secondary gene transfer with Ad-GFP control vector. Ten days after adenoviral gene transfer, the percentages of Treg were lowest in spleens of those mice that had been transduced with adenoviral vectors only, irrespective of the transgene. Finally, we wanted to test for induction of a Treg response in the liver, which is technically more challenging. Therefore, utilized FoxP3 reporter mice (FoxP3-IRES-EGFP knock-in mice) to + analyze for frequencies of Treg in form of EGFP intrahepatic lymphocytes 10 days after Ad-LacZ gene transfer. In AAV-LacZ pre-treated mice, the frequencies of Treg were significantly increased compared to AAV-LacZ transduced mice that did not receive the secondary gene transfer (Fig. 8C), while Treg were undetectable among hepatic lymphocytes from Ad-LacZ only treated mice. Hepatic gene transfer with AAV vectors has been shown to induce tolerance to a number of transgene products in animal models of human disease. This treatment provided immune tolerance to the therapeutic protein and, simultaneously, therapy or, alternatively, allowed for safe administration of supplementary therapy such as enzyme replacement therapy or therapeutic gene transfer to a different organ [35–37]. ...
Citations
... Resident antigen-presenting cells such as dendritic cells, Kupffer cells, liver sinusoidal endothelial cells, hepatocytes, and hepatic stellate cells present antigens in a tolerogenic manner to T cells and express immunosuppressive cytokines, resulting in regulatory T cells (Tregs) expansion, effector T cell anergy or death, and type 1 regulatory cell induction [40]. Likewise, AAV-mediated gene therapy targeting the liver has been shown to induce antigen-specific Tregs [41][42][43][44][45]. A rhesus macaque study observed no ADA to 4L6 mAb when the AAV8 vector was administered intravenously using a liver-specific TBG promoter. ...
Harnessing the immune system to combat disease has revolutionized medical treatment. Monoclonal antibodies (mAbs), in particular, have emerged as important immunotherapeutic agents with clinical relevance in treating a wide range of diseases, including allergies, autoimmune diseases, neurodegenerative disorders, cancer, and infectious diseases. These mAbs are developed from naturally occurring antibodies and target specific epitopes of single molecules, minimizing off-target effects. Antibodies can also be designed to target particular pathogens or modulate immune function by activating or suppressing certain pathways. Despite their benefit for patients, the production and administration of monoclonal antibody therapeutics are laborious, costly, and time-consuming. Administration often requires inpatient stays and repeated dosing to maintain therapeutic levels, limiting their use in underserved populations and developing countries. Researchers are developing alternate methods to deliver monoclonal antibodies, including synthetic nucleic acid-based delivery, to overcome these limitations. These methods allow for in vivo production of monoclonal antibodies, which would significantly reduce costs and simplify administration logistics. This review explores new methods for monoclonal antibody delivery, including synthetic nucleic acids, and their potential to increase the accessibility and utility of life-saving treatments for several diseases.
... 69,[79][80][81] Hepatocyte-derived expression of a secreted transgene protein may induce tolerance against this transgene protein, as demonstrated in mice. 69,82 This tolerance induction was mainly mediated by antigen-specific CD4 + CD25 + FoxP3 + regulatory T cells (Tregs). 82,83 Interestingly the CD8 + T cell-mediated clearance and induction of tolerance is dictated by dose levels. ...
... 69,82 This tolerance induction was mainly mediated by antigen-specific CD4 + CD25 + FoxP3 + regulatory T cells (Tregs). 82,83 Interestingly the CD8 + T cell-mediated clearance and induction of tolerance is dictated by dose levels. 66 A higher dose is thought to have higher immunogenicity risk, potentially leading to SAEs (Table 2). ...
Immunogenicity has imposed a challenge to efficacy and safety evaluation of adeno-associated virus (AAV) vector-based gene therapies. Mild to severe adverse events observed in clinical development have been implicated with host immune responses against AAV gene therapies, resulting in comprehensive evaluation of immunogenicity during nonclinical and clinical studies mandated by health authorities. Immunogenicity of AAV gene therapies is complex due to the number of risk factors associated with product components and pre-existing immunity in human subjects. Different clinical mitigation strategies have been employed to alleviate treatment-induced or -boosted immunogenicity in order to achieve desired efficacy, reduce toxicity, or treat more patients who are seropositive to AAV vectors. In this review, the immunogenicity risk assessment, manifestation of immunogenicity and its impact in nonclinical and clinical studies, and various clinical mitigation strategies are summarized. Last, we present bioanalytical strategies, methodologies, and assay validation applied to appropriately monitor immunogenicity in AAV gene therapy-treated subjects.
... 2.9.5. Inflammation of the liver parenchyma and portal ducts 0 ¼ none, 1 ¼ focal and mild lesions, 2 ¼ multifocal lesions, moderate, 3 ¼ multifocal lesions, severe, and 4 ¼ multifocal degeneration or necrosis [44]. ...
Paraquat (PQ) is a herbicide belonging to the group of bipyridylium salts. The objective of this study was to evaluate oxidative stress, DNA damage, and cytotoxicity induced by paraquat in peripheral lymphocyte cells in vivo as well as pathological changes in various tissues. For this purpose, 28 male Wistar rats in 6 different groups were poisoned by paraquat gavage and blood samples were taken from the hearts of rats after during the poisoning period. Oxidative stress, DNA damage, cell membrane integrity, serum lactate dehydrogenase, and cytotoxicity, were investigated by Ferric Reducing Antioxidant Potential (FRAP) test, alkaline comet assay, measuring serum lactate dehydrogenase (LDH), Hoechst staining and flow cytometry with propidium iodide (PI) respectively. The lung, kidney, and liver tissues were also examined pathologically. Paraquat caused dose-dependent DNA damage in peripheral lymphocyte cells and significant oxidative cell membrane damage. The most damage was caused by a single dose of 200 mg/kg b.w of paraquat by gavage. The gradual exposure to a dose of 300 mg/kg b.w of paraquat showed less damage, which could be due to the activation of the antioxidant defense mechanism.
... 44 AAV vectors have the potential to trigger transgene product-specific antibody responses especially upon i.m. injection, which is less prone than hepatic transfer to elicit immunosuppressive responses. 45 This would cause harm as it would affect traditional treatment by protein therapy. 46 Immunosuppression by steroids given alone or in combination with other drugs such as cyclosporin or mycophenolate mofetil is being used to block T cell responses to AAV-mediated gene transfer 2,47,48 ; their effect on B cell responses remains to be studied in more depth. ...
Adeno-associated virus (AAV) vector-mediated gene transfer is lessening the impact of monogenetic disorders. Human AAV gene therapy recipients commonly mount immune responses to AAV or the encoded therapeutic protein, which requires transient immunosuppression. Most efforts to date have focused on blunting AAV capsid-specific T cell responses, which have been implicated in elimination of AAV transduced cells. Here we explore the use of immunosuppressants, rapamycin given alone or in combination with ibrutinib to inhibit AAV vector- or transgene product-specific antibody responses. Our results show that rapamycin or ibrutinib given alone reduce primary antibody responses against AAV capsid but the combination of rapamycin and ibrutinib is more effective, blunts recall responses, and reduces numbers of circulating antibody-secreting plasma cells. The drugs fail to lower B cell memory formation or to reduce the inhibitory effects of pre-existing AAV capsid-specific antibodies on transduction efficiency.
... Importantly, transgene-specific immune responses following AAV gene transfer appear to be highly dependent on the target tissue and the route of virus administration. While AAV vector delivery to the liver seems to induce transgene tolerance in many cases [91,100,167], prolonged transgene expression following intramuscular AAV delivery required immunosuppression in several preclinical trials [42,48,51,154]. Furthermore, intramuscular administration of an AAV vector encoding microdystrophin to Duchenne muscular dystrophy patients also led to an immune response against the transgene [93]. ...
Paralysis is a frequent phenomenon in many diseases, and to date, only functional electrical stimulation (FES) mediated via the innervating nerve can be employed to restore skeletal muscle function in patients. Despite recent progress, FES has several technical limitations and significant side effects. Optogenetic stimulation has been proposed as an alternative, as it may circumvent some of the disadvantages of FES enabling cell type–specific, spatially and temporally precise stimulation of cells expressing light-gated ion channels, commonly Channelrhodopsin2. Two distinct approaches for the restoration of skeletal muscle function with optogenetics have been demonstrated: indirect optogenetic stimulation through the innervating nerve similar to FES and direct optogenetic stimulation of the skeletal muscle. Although both approaches show great promise, both have their limitations and there are several general hurdles that need to be overcome for their translation into clinics. These include successful gene transfer, sustained optogenetic protein expression, and the creation of optically active implantable devices. Herein, a comprehensive summary of the underlying mechanisms of electrical and optogenetic approaches is provided. With this knowledge in mind, we substantiate a detailed discussion of the advantages and limitations of each method. Furthermore, the obstacles in the way of clinical translation of optogenetic stimulation are discussed, and suggestions on how they could be overcome are provided. Finally, four specific examples of pathologies demanding novel therapeutic measures are discussed with a focus on the likelihood of direct versus indirect optogenetic stimulation.
... 104 Several approaches have been tested in mice to limit transgene product immunity following IM AAV gene delivery including vector genome engineering to remove TLR9 stimulatory CpGs, 84 tissue-specific promoters and hematopoietic lineage microRNA (miRNA) targets 142-3p, 105 transient immune modulation, 40,106 and dual expression of transgene in liver and muscle. [107][108][109][110] Studies in large animal models have shown that the delivery route, direct IM injection versus regional vascular delivery, also impacts transgene immunity. 111 A recently published study reports 5-year inducible expression of the immunogenic doxycycline Tet-On system in NHPs following local regional delivery of an AAV vector with no detectable transgene product immune responses. ...
... Liver transduction by an AAV2-LacZ vector prior to an Ad-LacZ vector resulted in sustained expression and suppression of transgene-specific CD8 + T cell activation that was seen in Ad-LacZ only treated controls. 107 Expression of a membrane-bound ova protein in hepatocytes was shown to prevent airway-induced allergy mediated by Treg induction. 125 And finally, administration of an AAV8-MOG vector was shown to prevent and reverse disease in experimental autoimmune encephalomyelitis (EAE) mice, a model for multiple sclerosis. ...
Early preclinical studies in rodents and other species did not reveal that vector or transgene immunity would present a significant hurdle for sustained gene expression. While there was early evidence of mild immune responses to adeno-associated virus (AAV) in preclinical studies, it was generally believed that these responses were too weak and transient to negatively impact sustained transduction. However, translation of the cumulative success in treating hemophilia B in rodents and dogs with an AAV2-F9 vector to human studies was not as successful. Despite significant progress in recent clinical trials for hemophilia, new immunotoxicities to AAV and transgene are emerging in humans that require better animal models to assess and overcome these responses. The animal models designed to address these immune complications have provided critical information to assess how vector dose, vector capsid processing, vector genome, difference in serotypes, and variations in vector delivery route can impact immunity and to develop approaches for overcoming pre-existing immunity. Additionally, a comprehensive dissection of innate, adaptive, and regulatory responses to AAV vectors in preclinical studies has provided a framework that can be utilized for development of immunomodulatory therapies to overcome or bypass immune responses and for developing strategic approaches toward engineering stealth AAV vectors that can circumvent immunity.
... It is possible to induce tolerance to GAA by pretreating with hepatic AAV-GAA gene transfer which prevents the predisposition to anaphylactic reactions (100). These observations are based on the pioneering work by Herzog et al., where hepatocyte-derived expression induces transgene product-specific immune tolerance (101)(102)(103) in which regulatory T-cells actively suppress B and T cells (102,104,105). Additional approaches toward immune tolerance induction have been developed using immune suppressive drugs such as rapamycin in combination with B cell-depleting antibodies (36,50,101). ...
Pompe disease is a neuromuscular disease caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase leading to lysosomal and cytoplasmic glycogen accumulation in neurons and striated muscle. In the decade since availability of first-generation enzyme replacement therapy (ERT) a better understanding of the clinical spectrum of disease has emerged. The most severe form of early onset disease is typically identified with symptoms in the first year of life, known as infantile-onset Pompe disease (IOPD). Infants are described at floppy babies with cardiac hypertrophy in the first few months of life. A milder form with late onset (LOPD) of symptoms is mostly free of cardiac involvement with slower rate of progression. Glycogen accumulation in the CNS and skeletal muscle is observed in both IOPD and LOPD. In both circumstances, multi-system disease (principally motoneuron and myopathy) leads to progressive weakness with associated respiratory and feeding difficulty. In IOPD the untreated natural history leads to cardiorespiratory failure and death in the first year of life. In the current era of ERT clinical outcomes are improved, yet, many patients have an incomplete response and a substantial unmet need remains. Since the neurological manifestations of the disease are not amenable to peripheral enzyme replacement, we set out to better understand the pathophysiology and potential for treatment of disease manifestations using adeno-associated virus (AAV)-mediated gene transfer, with the first clinical gene therapy studies initiated by our group in 2006. This review focuses on the preclinical studies and clinical study findings which are pertinent to the development of a comprehensive gene therapy strategy for both IOPD and LOPD. Given the advent of newborn screening, a significant focus of our recent work has been to establish the basis for repeat administration of AAV vectors to enhance neuromuscular therapeutic efficacy over the life span.
... 86 The lack of responsiveness observed in liver-directed gene transfer with AAV vectors appears to be mediated by antigen-specific CD4 + CD25 + FoxP3 + Tregs, 53 which play a central role in liver-mediated tolerance induction. 52,54 Importantly, liver-mediated tolerance can be induced for various transgenes 53,87,88 and can be used to eradicate ongoing antibody responses to antigens. [89][90][91][92][93] The development of detrimental antibody responses to therapeutic proteins is a common complication encountered in patients affected by recessive diseases who are treated by protein replacement therapy. ...
In recent years, the number of clinical trials in which adeno-associated virus (AAV) vectors have been used for in vivo gene transfer has steadily increased. The excellent safety profile, together with the high efficiency of transduction of a broad range of target tissues, has established AAV vectors as the platform of choice for in vivo gene therapy. Successful application of the AAV technology has also been achieved in the clinic for a variety of conditions, including coagulation disorders, inherited blindness, and neurodegenerative diseases, among others. Clinical translation of novel and effective "therapeutic products" is, however, a long process that involves several cycles of iterations from bench to bedside that are required to address issues encountered during drug development. For the AAV vector gene transfer technology, several hurdles have emerged in both preclinical studies and clinical trials; addressing these issues will allow in the future to expand the scope of AAV gene transfer as a therapeutic modality for a variety of human diseases. In this review, we will give an overview on the biology of AAV vector, discuss the design of AAV-based gene therapy strategies for in vivo applications, and present key achievements and emerging issues in the field. We will use the liver as a model target tissue for gene transfer based on the large amount of data available from preclinical and clinical studies.
... Tregs that modulate immune tolerance [6,8,14,[28][29][30][31]. To date, most studies supporting liver induced tolerance have utilized mouse models, especially when focusing on the mechanisms behind the induction of tolerance. ...
Immune tolerance is a vital component of immunity, as persistent activation of immune cells causes significant tissue damage and loss of tolerance leads to autoimmunity. Likewise, unwanted immune responses can occur in inherited disorders, such as hemophilia and Pompe disease, in which patients lack any expression of protein, during treatment with enzyme replacement therapy, or gene therapy. While the liver has long been known as being tolerogenic, it was only recently appreciated in the last decade that liver directed adeno-associated virus (AAV) gene therapy can induce systemic tolerance to a transgene. In this review, we look at the mechanisms behind liver induced tolerance, discuss different factors influencing successful tolerance induction with AAV, and applications where AAV mediated tolerance may be helpful.
... Several groups showed that delivery of rAAV vectors to the liver induces transgene-specific tolerance. [105][106][107][108][109] Accordingly, rAAV-mediated liver gene transfer has also been used in inhibitor-prone hemophilia A dogs to eradicate low-titer anti-FVIII neutralizing antibodies, 110 and in mice to eradicate antibodies to FIX 111 or alpha-acid glucosidase. 112 Although these preclinical data on liver tolerance are highly convincing, the open question is whether this concept will reliably translate to humans. ...
Achromatopsia is an inherited retinal disorder of cone photoreceptors characterized by markedly reduced visual acuity, extreme light sensitivity and absence of color discrimination. Approximately 50% of cases are caused by mutations in the cone photoreceptor-specific cyclic nucleotide gated channel beta subunit (CNGB3) gene. Studies in CNGB3-mutant dogs showed that subretinal injection of an AAV vector expressing human CNGB3, which has 76% amino acid identity with canine CNGB3, driven by a 2.1 kb human red cone opsin promoter (PR2.1) and packaged in AAV5 capsids (AAV5-PR2.1-hCNGB3) rescued cone photoreceptor function, but at high doses was associated with an inflammatory response (focal chorioretinitis) consistent with immune-mediated toxicity. AAV vectors containing the PR2.1 promoter packaged in AAV5 capsids and expressing either the native canine CNGB3 (AAV5-PR2.1-cCNGB3) or the human CNGB3 (AAV5-PR2.1-hCNGB3) were evaluated at different dose levels in CNGB3-mutant dogs. The vector expressing canine CNGB3 achieved somewhat better rescue of cone function but unexpectedly was associated with a greater degree of retinal toxicity than the vector expressing human CNGB3. Very low level T cell immune responses to some AAV or CNGB3 peptides were observed in animals that received the higher vector dose. There was a >2-fold increase in serum neutralizing antibodies to AAV in 1 of 3 animals in the low dose group and 2 of 3 animals in the high dose group. No serum anti-hCNGB3 antibodies were detected in any animal. Results of this study do not support the hypothesis that the focal chorioretinitis seen with high doses of AAV5-PR2.1-hCNGB3 in the initial studies was due to an immune response to human CNGB3.
