Vector-treated Mice Research Articles

The fetal mouse is a sensitive genotoxicity model that exposes lentiviral-associated mutagenesis resulting in liver oncogenesis.

Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.

Targeting nicotine

The development of an effective anti-nicotine vaccine to aid smoking cessation, by preventing nicotine from entering the brain, has been hampered by the inability of the tested vaccines to generate consistently high antibody titres in humans. Now, Hicks et al. have created an adeno-associated virus gene transfer vector that is capable of expressing high levels of a monoclonal anti-nicotine antibody. Following systemic nicotine administration, vector-treated mice had markedly lower levels of brain nicotine than did untreated mice, and treatment also prevented nicotine-induced cardiovascular and locomotor effects. Thus, passive anti-nicotine immunotherapy may warrant further study as a possible anti-smoking therapy.

Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy

Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 1012 vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD−/− mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD−/− mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD−/− mice maintained euglycemia, whereas untreated VLCAD−/− mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.

Foamy Virus Vector-mediated Gene Correction of a Mouse Model of Wiskott–Aldrich Syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by eczema, thrombocytopenia and immunodeficiency. Hematopoietic cell transplantation can cure the disease and gene therapy is being tested as an alternative treatment option. In this study, we assessed the use of foamy virus (FV) vectors as a gene transfer system for WAS, using a Was knockout (KO) mouse model. Preliminary experiments using FV vectors expressing the green fluorescent protein under the transcriptional control of the endogenous WAS promoter or a ubiquitously acting chromatin opening element allowed us to define transduction conditions resulting in high (>40%) and long-term in-vivo marking of blood cells after transplantation. In following experiments, Was KO mice were treated with FV vectors containing the human WAS complementary DNA (cDNA). Transplanted animals expressed the WAS protein (WASp) in T and B lymphocytes, as well as platelets and showed restoration of both T-cell receptor-mediated responses and B-cell migration. We also observed recovery of platelet adhesion and podosome formation in dendritic cells (DCs) of treated mice. These data demonstrate that FV vectors can be effective for hematopoietic stem cell (HSC)-directed gene correction of WAS.

Abstract P138: PTEN Mediates Post--Myocardial Infarction Remodeling

Background- Following myocardial infarction (MI), innate immunity is activated, leading to cytokine production and initiation of inflammation. Interleukin (IL)-10 has been reported to attenuate post-MI remodeling by inhibiting expression of tumor necrosis factor (TNF)-α. The phosphatase and tensin homologue deleted on chromosome ten (PTEN) plays an important role in myocardial ischemia-reperfusion injury. It is not known whether PTEN regulates cytokine production and cardiac remodeling. Here, we have investigated the hypothesis that PTEN mediates post-MI remodeling by inhibiting IL-10. Methods and Results- MI was induced in wildtype (WT) mice and Pten heterozygous (HET) mice. In a separate experiment, Pten adenoviruses or empty viruses were injected into the peri-infarct area of WT mice. Left ventricular (LV) anatomical and functional changes were assessed by echocardiography. Mononuclear cell infiltration was measured by histology. TNF-α, IL-10, matrix metalloproteinase (MMP)-2 and MMP-9 protein levels were analyzed in the peri-infarct area. At the end of the experiments, LV end-diastolic diameter (LVEDD) was decreased and fractional shortening (FS) was increased in HET mice compared with WT mice (LVEDD: 4.13±0.22 vs. 5.42±0.26 mm, p<0.01; FS: 21.9±2.6 vs. 11.5±1.1 %, p<0.001). Moreover, heart weight and infarct size were decreased in HET mice compared with WT mice. However, FS was decreased and LV rupture (LVR) was increased in Pten adenovirus-treated mice compared with empty vector-treated mice [FS: 8.2±1.1 vs. 21.5±3.2 %, p < 0.01; LVR: 100% (16/16) vs. 33% (3/9)]. Mononuclear cell infiltration was attenuated in HET mice and worsened in Pten adenovirus-treated mice. IL-10 protein levels were upregulated and TNF-α, MMP-2, and MMP-9 protein levels were downregulated in HET mice. The opposite effects were found in Pten over-expressing hearts. Conclusions- PTEN downregulation increases IL-10 production and inhibits inflammatory responses and attenuates cardiac remodeling; conversely, PTEN over-expression inhibits IL-10 expression and increases cardiac injury. Therefore, our studies demonstrate that PTEN mediates post-MI remodeling, at least in part, by regulating IL-10 production.

Hepatic Gene Transfer of Factor IX Reverses Inhibitors and Protects From Anaphylaxis in a Murine Hemophilia B Model

Abstract 669▪▪This icon denotes a clinically relevant abstractHemophilia B is a X-linked bleeding disorder caused by loss of coagulation factor IX (F.IX) function. Under current treatment protocols, approximately 2–4% of hemophilia B patients develop inhibitory antibodies to F.IX protein, and those with F.IX gene deletions are at risk for anaphylaxis. We work with a murine model for this pathogenic antibody response, namely C3H/HeJ hemophilia B (HB) mice with a F9 gene deletion. Anaphylactic reactions to intravenously administered F.IX protein are the result of a strong Th2-driven antibody response, comprised of IgG1 and IgE. Interestingly, we find that IgE levels correlate with the dose of recombinant F.IX protein. HB mice treated with weekly intravenous doses of 0.1 or 0.3 IU F.IX were free from allergic/anaphylactic reactions during the course of treatment (1 IP and 5 weekly IV injections of F.IX protein) and had very low to undetectable IgE against F.IX. In contrast, a dose of 1 IU induced IgE formation and caused fatal anaphylactic reactions after repeated administration. These results suggest that prophylaxis with lower F.IX doses may be recommended in individuals with F.IX gene deletion (Mice with missense or nonsense F.IX mutations did not show inhibitor formation or anaphylaxis at the high dose). In an attempt to reverse the response and desensitize the gene deletion HB animals, F.IX-treated mice with inhibitors, including those with detectable circulating IgE, received hepatic AAV8-F.IX gene transfer (1e11 vg/mouse). Within one month, these mice lost detectable IgG and IgE against F.IX and showed a level of correction of coagulation comparable to gene transfer in naive mice. Subsequent intravenous injections of 1 IU F.IX (weekly for 1 month) did not cause anaphylaxis, demonstrating successful desensitization. Control mice (no gene transfer) maintained their IgE levels during the course of the experiment. We are currently investigating if a lower AAV8 F.IX vector dose (1e10 vg) is similarly capable of reversing existing F.IX inhibitors/anaphylaxis. In order to determine the fate of F.IX antibody-secreting cells (ASC), we performed a B cell ELISPOT assay on bone marrow cells and splenocytes in F.IX immunized HB mice left untreated or treated with AAV8 F.IX vector. Control immunized mice showed predominantly ASC in the spleen with a few detected cells from bone marrow. ASC were nearly undetectable in vector-treated mice. These results suggest that AAV8 liver gene transfer not only suppresses F.IX-specific ASC, but may also prevent the re-activation of memory B cells. Experiments are ongoing to explore the role of induced regulatory T cells in modulating ASC. To characterize T cell responses against F.IX, we isolated splenocytes isolated from control immunized and vector treated mice, stimulated these cells in vitro with F.IX protein, and collected RNA for analysis with RT-PCR array. Control mice showed an upregulation of Th2 cytokines IL-4 and IL-13, which are known to induce B cell class switching to IgG1 and to IgE, corresponding to the observed antibody formation. Vector treated immunized mice did not show up regulation of any cytokines representative of Th1 or Th2 responses, indicating down-regulation of T help required for the antibody response. In summary, our data show that liver directed AAV8 F.IX gene transfer may not only induce tolerance to those at risk of developing inhibitors, but may offer an alternative treatment approach to expensive and long-term immune tolerance induction (ITI) protocols in those with existing pathogenic antibody responses. This concept may also apply to other genetic diseases, in which antibodies complicate protein replacement therapy. Disclosures:Herzog:Genzyme Corporation: Patents & Royalties.

Local Delivery of GM-CSF Protects Mice from Lethal Pneumococcal Pneumonia

The growth factor GM-CSF has an important role in pulmonary surfactant metabolism and the regulation of antibacterial activities of lung sentinel cells. However, the potential of intra-alveolar GM-CSF to augment lung protective immunity against inhaled bacterial pathogens has not been defined in preclinical infection models. We hypothesized that transient overexpression of GM-CSF in the lungs of mice by adenoviral gene transfer (Ad-GM-CSF) would protect mice from subsequent lethal pneumococcal pneumonia. Our data show that intra-alveolar delivery of Ad-GM-CSF led to sustained increased pSTAT5 expression and PU.1 protein expression in alveolar macrophages during a 28-d observation period. Pulmonary Ad-GM-CSF delivery 2-4 wk prior to infection of mice with Streptococcus pneumoniae significantly reduced mortality rates relative to control vector-treated mice. This increased survival was accompanied by increased inducible NO synthase expression, antibacterial activity, and a significant reduction in caspase-3-dependent apoptosis and secondary necrosis of lung sentinel cells. Importantly, therapeutic treatment of mice with rGM-CSF improved lung protective immunity and accelerated bacterial clearance after pneumococcal challenge. We conclude that prophylactic delivery of GM-CSF triggers long-lasting immunostimulatory effects in the lung in vivo and rescues mice from lethal pneumococcal pneumonia by improving antibacterial immunity. These data support use of novel antibiotic-independent immunostimulatory therapies to protect patients against bacterial pneumonias.

Hepatorenal Correction in Murine Glycogen Storage Disease Type I With a Double-stranded Adeno-associated Virus Vector

Glycogen storage disease type Ia (GSD-Ia) is caused by the deficiency of glucose-6-phosphatase (G6Pase). Long-term complications of GSD-Ia include life-threatening hypoglycemia and proteinuria progressing to renal failure. A double-stranded (ds) adeno-associated virus serotype 2 (AAV2) vector encoding human G6Pase was pseudotyped with four serotypes, AAV2, AAV7, AAV8, and AAV9, and we evaluated efficacy in 12-day-old G6pase (-/-) mice. Hypoglycemia during fasting (plasma glucose <100 mg/dl) was prevented for >6 months by the dsAAV2/7, dsAAV2/8, and dsAAV2/9 vectors. Prolonged fasting for 8 hours revealed normalization of blood glucose following dsAAV2/9 vector administration at the higher dose. The glycogen content of kidney was reduced by >65% with both the dsAAV2/7 and dsAAV2/9 vectors, and renal glycogen content was stably reduced between 7 and 12 months of age for the dsAAV2/9 vector-treated mice. Every vector-treated group had significantly reduced glycogen content in the liver, in comparison with untreated G6pase (-/-) mice. G6Pase was expressed in many renal epithelial cells of with the dsAAV2/9 vector for up to 12 months. Albuminuria and renal fibrosis were reduced by the dsAAV2/9 vector. Hepatorenal correction in G6pase (-/-) mice demonstrates the potential of AAV vectors for the correction of inherited diseases of metabolism.

Decoy receptor 3 inhibits renal mononuclear leukocyte infiltration and apoptosis and prevents progression of IgA nephropathy in mice

The progression of IgA nephropathy (IgAN), the most frequent type of primary glomerulonephritis, is associated with high levels of mononuclear leukocyte infiltration into the kidney. These cells consist mainly of T cells and macrophages. Our previous study showed that a decoy receptor 3 (DCR3) gene therapy can prevent the development of a mouse autoimmune glomerulonephritis model by its potent immune modulating effects (Ka SM, Sytwu HK, Chang DM, Hsieh SL, Tsai PY, Chen A. J Am Soc Nephrol 18: 2473-2485, 2007). Here, we tested the hypothesis that DCR3 might prevent the progression of IgAN, an immune complex-mediated primary glomerulonephritis, by inhibiting T cell activation, renal T cell/macrophage infiltration, and protecting the kidney from apoptosis. We used a progressive IgAN (Prg-IgAN) model in B cell-deficient mice, because the mice are characterized by a dramatic proliferation of activated T cells systemically and progressive NF-κB activation in the kidney. We treated the animals with short-term gene therapy with DCR3 plasmids by hydrodynamics-based gene delivery. When the mice were euthanized on day 21, we found that, compared with empty vector-treated (disease control) Prg-IgAN mice, DCR3 gene therapy resulted in 1) systemic inhibition of T cell activation and proliferation; 2) lower serum levels of proinflammatory cytokines; 3) improved proteinuria, renal function, and renal pathology (inhibiting the development of marked glomerular proliferation, crescent formation, glomerulosclerosis, and interstitial inflammation); 5) suppression of T cell and macrophage infiltration into the periglomerular interstitium of the kidney; and 5) a reduction in apoptotic figures in the kidney. On the basis of these findings, DCR3 might be useful therapeutically in preventing the progression of IgAN.

Thrombospondin-1 Short Hairpin RNA Suppresses Tubulointerstitial Fibrosis in the Kidney of Ureteral Obstruction by Ameliorating Peritubular Capillary Injury

Background/Aims: Thrombospondin-1 (TSP-1), a naturally occurring inhibitor of angiogenesis, is an important mediator of renal fibrosis in clinical and experimental kidney disease. Increasing evidence shows that the microvasculature plays a critical role in progressive renal disease. The present study was undertaken to investigate whether interstitial fibrosis could be prevented by abolishing TSP-1 function in unilateral ureteral obstruction (UUO)-induced renal fibrosis. Methods and Results: A short hairpin RNA vector, designated Thbs-1, significantly suppressed TSP-1 in both transcriptional and translational levels in in vitro-cultured cells and in vivo fibrosis-induced mouse kidney. Furthermore, TSP-1 RNA interference increased the protein level of vascular endothelial growth factor (VEGF) and the density of peritubular capillaries (PTCs), reduced the expression of hypoxia-inducible factor-1α in tubulointerstitial cells, and collagen III and the connective tissue growth factor expression were markedly reduced from day 7 after UUO-induced fibrosis, but un- or vector-treated mice maintained their expression. TSP-1 shRNA suppressed the protein level of TSP-1, increased VEGF expression and PTC density and alleviated the development of renal interstitial fibrosis in UUO mice. Conclusion: These data suggest that inhibition of TSP-1 expression prevented tubulointerstitial fibrosis through ameliorating PTC injury.

Induction and Prevention of Severe Hyperammonemia in the spfash Mouse Model of Ornithine Transcarbamylase Deficiency Using shRNA and rAAV-mediated Gene Delivery

Urea cycle defects presenting early in life with hyperammonemia remain difficult to treat and commonly necessitate liver transplantation. Gene therapy has the potential to prevent hyperammonemic episodes while awaiting liver transplantation, and possibly also to avert the need for transplantation altogether. Ornithine transcarbamylase (OTC) deficiency, the most prevalent urea cycle disorder, provides an ideal model for the development of liver-targeted gene therapy. While we and others have successfully cured the spf(ash) mouse model of OTC deficiency using adeno-associated virus (AAV) vectors, a major limitation of this model is the presence of residual OTC enzymatic activity which confers a mild phenotype without clinically significant hyperammonemia. To better model severe disease we devised a strategy involving AAV2/8-mediated delivery of a short hairpin RNA (shRNA) to specifically knockdown residual endogenous OTC messenger RNA (mRNA). This strategy proved highly successful with vector-treated mice developing severe hyperammonemia and associated neurological impairment. Using this system, we showed that the dose of an AAV rescue construct encoding the murine OTC (mOTC) cDNA required to prevent hyperammonemia is fivefold lower than that required to control orotic aciduria. This result is favorable for clinical translation as it indicates that the threshold for therapeutic benefit is likely to be lower than indicated by earlier studies.

Effects of irradiating adult mdx mice before full-length dystrophin cDNA transfer on host anti-dystrophin immunity

Duchenne muscular dystrophy is a fatal, genetic disorder in which dystrophin-deficient muscle progressively degenerates, for which dystrophin gene transfer could provide effective treatment. The host immune response to dystrophin, however, is an obstacle to therapeutic gene expression. Understanding the dystrophin-induced host immune response will facilitate the discovery of strategies to prolong expression of recombinant dystrophin in dystrophic muscle. Using whole-body irradiation of the dystrophic mdx mouse before gene transfer, we temporally removed the immune system; a 600 rad dose removed peripheral immune cells, which were restored by self-reconstitution, and a 900 rad dose removed central and peripheral immune cells, which were restored by adoptive transfer of bone marrow from a syngeneic, dystrophin-normal donor. The anti-dystrophin humoral response was delayed and dystrophin expression was partially preserved in irradiated, vector-treated mice. Nonirradiated, vector-treated control mice lost muscle dystrophin expression completely, had an earlier anti-dystrophin humoral response and demonstrated muscle fibers focally surrounded with T cells. We conclude that dystrophin gene transfer induced anti-dystrophin humoral immunity and cell-mediated responses that were significantly diminished and delayed by temporal removal of the host central or peripheral immune cells. Furthermore, manipulation of central immunity altered the pattern of regulatory T cells in muscle.

Sexually Dimorphic Patterns of Episomal rAAV Genome Persistence in the Adult Mouse Liver and Correlation With Hepatocellular Proliferation

Recombinant adeno-associated virus vectors (rAAVs) show exceptional promise for liver-targeted gene therapy, with phenotype correction in small and large animal disease models being reported with increasing frequency. Success in humans, however, remains a considerable challenge that demands greater understanding of host-vector interactions, notably those governing the efficiency of initial gene transfer and subsequent long-term persistence of gene expression. In this study, we examined long-term enhanced green fluorescent protein (eGFP) expression and vector genome persistence in the mouse liver after rAAV2/8-mediated gene transfer in early adulthood. Two intriguing findings emerged of considerable scientific and clinical interest. First, adult female and male mice showed distinctly different patterns of persistence of eGFP expression across the hepatic lobule after exhibiting similar patterns initially. Female mice retained a predominantly perivenous pattern of expression, whereas male mice underwent inversion of this pattern with preferential loss of perivenous expression and relative retention of periportal expression. Second, these changing patterns of expression correlated with sexually dimorphic patterns of genome persistence that appear linked both spatially and temporally to underlying hepatocellular proliferation. Observation of the equivalent phenomenon in man could have significant implications for the long-term therapeutic efficacy of rAAV-mediated gene transfer, particularly in the context of correction of liver functions showing metabolic zonation.

Scavenging superoxide selectively in mouse forebrain is associated with improved cardiac function and survival following myocardial infarction

Dysregulation in central nervous system (CNS) signaling that results in chronic sympathetic hyperactivity is now recognized to play a critical role in the pathogenesis of heart failure (HF) following myocardial infarction (MI). We recently demonstrated that adenovirus-mediated gene transfer of cytoplasmic superoxide dismutase (Ad-Cu/ZnSOD) to forebrain circumventricular organs, unique sensory structures that lack a blood-brain barrier and link peripheral blood-borne signals to central nervous system cardiovascular circuits, inhibits both the MI-induced activation of these central signaling pathways and the accompanying sympathoexcitation. Here, we tested the hypothesis that this forebrain-targeted reduction in oxidative stress translates into amelioration of the post-MI decline in myocardial function and increase in mortality. Adult C57BL/6 mice underwent left coronary artery ligation or sham surgery along with forebrain-targeted gene transfer of Ad-Cu/ZnSOD or a control vector. The results demonstrate marked MI-induced increases in superoxide radical formation in one of these forebrain regions, the subfornical organ (SFO). Ad-Cu/ZnSOD targeted to this region abolished the increased superoxide levels and led to significantly improved myocardial function compared with control vector-treated mice. This was accompanied by diminished levels of cardiomyocyte apoptosis in the Ad-Cu/ZnSOD but not the control vector-treated group. These effects of superoxide scavenging with Ad-Cu/ZnSOD in the forebrain paralleled increased post-MI survival rates compared with controls. This suggests that oxidative stress in the SFO plays a critical role in the deterioration of cardiac function following MI and underscores the promise of CNS-targeted antioxidant therapy for the treatment of MI-induced HF.

Abstract 3810: Antiviral RNA Interference Mediated by a Recombinant Cardiotropic AAV9 Vector Improves Cardiac Function in Coxsackievirus B3 Cardiomyopathy

RNA interference (RNAi) has potential to be a novel therapeutic strategy in diverse areas of medicine. We report here on targeted RNAi for the treatment of a viral cardiomyopathy which is a major cause of sudden cardiac death or terminal heart failure in children and young adults. RNAi therapy employs small regulatory RNAs to achieve its effect but in vivo use of synthetic small interfering RNAs is limited by instability in plasma and low transfer into target cells. We instead evaluated an RNAi strategy using short hairpin RNA (shRdRp) directed at the RNA polymerase (RdRP) of Coxsackievirus B3 (CoxB3) in HeLa cells, primary rat cardiomyocytes (PNCMs), and CoxB3-infected mice in vivo. A conventional AAV2 vector expressing shRdRp protected HeLa against virus-induced death, but this vector type was unable to transduce PNCMs. In contrast, an analogous pseudotyped AAV2.6 vector was protective also in PNCMs and reduced virus replication by &gt;3 log10 steps. Finally, we evaluated intravenous treatment of mice with an AAV2.9-shRdRp vector since AAV9 carries the most cardiotropic AAV capsid currently known for in vivo use. Mice with CoxB3 cardiomyopathy had disturbed left ventricular (LV) function with impaired parameters of contractility (dP/dtmax 3006±287 vs. 7482±487 mmHg/s, p&lt;0.01) and diastolic relaxation (dP/dtmin -2224±195 vs. -6456±356 mmHg/s, p&lt;0.01 and Tau 16.2±1.1 vs. 10.7±0.6 ms, p&lt;0.01) as compared to control mice. AAV2.9-shRdRp treatment significantly attenuated the cardiac dysfunction compared to control vector-treated mice on day 10 after CoxB3 infection: dP/dtmax 3865±354 vs. 3006±287 mmHg/s (p&lt;0.05) and dP/dtmin -3245±231 vs. −2224±195, mmHg/s (p&lt;0.05), and Tau 11.9±0.5 vs. 16.2±1.1 ms (p&lt;0.01). The data show, for the first time, that intravenously injected AAV9 has the potential to target RNAi to the heart and suggest AAV9-shRNA vectors as a novel therapeutic approach for cardiac disorders.

Recombinant adeno‐associated virus‐mediated gene delivery of long chain acyl coenzyme A dehydrogenase (LCAD) into LCAD‐deficient mice

Very long chain acyl coenzyme A (CoA) dehydrogenase (VLCAD) deficiency is a relatively common mitochondrial beta-oxidation disorder. The most severe form of VLCAD deficiency presents with neonatal cardiomyopathy and hepatic failure and is generally fatal within the first year of life. Mice deficient for long chain acyl CoA dehydrogenase (LCAD) closely resemble the clinical syndrome observed in VLCAD-deficient humans. Recombinant adeno-associated viral (rAAV) vectors with pseudotype capsids were investigated for their potential towards correcting the phenotype observed in mice heterozygous (+/-) for LCAD (i.e. liver and muscle steatosis). rAAV containing the mouse LCAD cDNA (mLCAD) under the transcriptional control of the CMV/chicken beta-actin hybrid promoter were injected intramuscularly into the tibialis anterior (TA) muscle of LCAD(+/-) mice or injected into the portal vein to transduce hepatocytes. Ten weeks post-injection of rAAV1-mLCAD into the TA muscle, significantly increased levels of mLCAD within mitochondria were demonstrated by immunostaining of TA sections, immunoblotting of mitochondrial isolates and by the electron transfer flavoprotein (ETF) fluorescence reduction enzyme activity assay. Magnetic resonance spectroscopy of vector-injected TA muscle demonstrated a reduction in the lipid content compared to phosphate-buffered saline-injected mice, whereas a systemic effect was observed as a reduction in liver macrosteatosis. Eight weeks after portal vein injection of rAAV8-mLCAD into LCAD(+/-) mice, increased levels of mLCAD within hepatocyte mitochondria were demonstrated by immunostaining and also by the ETF assay. Scoring of the hepatosteatosis observed in partially deficient LCAD mice indicated a reduction in the lipid content within livers of vector-treated mice. These studies show that rAAV-mediated delivery of mLCAD was efficient and led to an amelioration of local and systemic pathologies observed in partially deficient LCAD mice.

Cardiac-targeted RNA interference mediated by an AAV9 vector improves cardiac function in coxsackievirus B3 cardiomyopathy

RNA interference (RNAi) has potential to be a novel therapeutic strategy in diverse areas of medicine. In this paper, we report on targeted RNAi for the treatment of a viral cardiomyopathy, which is a major cause of sudden cardiac death or terminal heart failure in children and young adults. RNAi therapy employs small regulatory RNAs to achieve its effect, but in vivo use of synthetic small interfering RNAs is limited by instability in plasma and low transfer into target cells. We instead evaluated an RNAi strategy using short hairpin RNA (shRdRp) directed at the RNA polymerase (RdRP) of coxsackievirus B3 (CoxB3) in HeLa cells, primary rat cardiomyocytes (PNCMs) and CoxB3-infected mice in vivo. A conventional AAV2 vector expressing shRdRp protected HeLa against virus-induced death, but this vector type was unable to transduce PNCMs. In contrast, an analogous pseudotyped AAV2.6 vector was protective also in PNCMs and reduced virus replication by >3 log10 steps. Finally, we evaluated the intravenous treatment of mice with an AAV2.9-shRdRp vector because AAV9 carries the most cardiotropic AAV capsid currently known for in vivo use. Mice with CoxB3 cardiomyopathy had disturbed left ventricular (LV) function with impaired parameters of contractility (dP/dtmax = 3,006 +/- 287 vs. 7,482 +/- 487 mmHg/s, p < 0.01) and diastolic relaxation (dP/dtmin = -2,224 +/- 195 vs. -6,456 +/- 356 mmHg/s, p < 0.01 and Tau = 16.2 +/- 1.1 vs. 10.7 +/- 0.6 ms, p < 0.01) compared to control mice. AAV2.9-shRdRp treatment significantly attenuated the cardiac dysfunction compared to control vector-treated mice on day 10 after CoxB3 infection: dP/dtmax = 3,865 +/- 354 vs. 3,006 +/- 287 mmHg/s (p < 0.05), dP/dtmin = -3,245 +/- 231 vs. -2,224 +/- 195 mmHg/s (p < 0.05) and Tau = 11.9 +/- 0.5 vs. 16.2 +/- 1.1 ms (p < 0.01). The data show, for the first time, that intravenously injected AAV9 has the potential to target RNAi to the heart and suggest AAV9-shRNA vectors as a novel therapeutic approach for cardiac disorders.

Adenoviral-vector mediated transfer of HBV-targeted ribonuclease can inhibit HBV replication in vivo

Hepatitis B virus (HBV)-targeted ribonuclease (HBV-TR) is a fused protein of HBV core protein and a ribonuclease, human eosinophil-derived neurotoxin (hEDN). Our previous results showed that HBV-TR could effectively inhibit HBV replication in vitro. To test whether HBV-TR can inhibit HBV replication in vivo, we constructed a recombinant adenoviral vector expressing HBV-TR (Ad-TR) and used it to treat HBV-transgenic mice. Immunohistochemical staining showed that TR was expressed at varied levels in different tissues of Ad-TR-treated mice. Serum HBsAg concentration was decreased by 64.8% for the Ad-TR-treated mice compared with empty adenoviral vector-treated control mice. The amount of HBV-DNA in the livers of the Ad-TR-treated mice was 0.74 × 10 7 copies/μg of genomic DNA while the amount of HBV-DNA in the livers of the empty adenoviral vector-treated control mice was 2.86 × 10 7 copies/μg of genomic DNA. Serum HBV-DNA of Ad-TR-treated mice was also decreased by 71.4% compared with empty adenoviral vector-treated control mice. In addition, for some Ad-TR-treated mice, the expression of HBsAg in the liver cells turned negative. No discernible adverse effects were observed for Ad-TR-treated mice. Taken together, our results indicated that adenovirus mediated transfer of HBV-TR can inhibit HBV replication in vivo.

871. Complete Restoration of In Vivo Oxidative Capacity for Phenylalanine in Self-Complementary AAV Vector-Treated Phenylketonuria Mice

871. Complete Restoration of In Vivo Oxidative Capacity for Phenylalanine in Self-Complementary AAV Vector-Treated Phenylketonuria Mice

AAV Vector-mediated Reversal of Hypoglycemia in Canine and Murine Glycogen Storage Disease Type Ia

Glycogen storage disease type Ia (GSD-Ia) profoundly impairs glucose release by the liver due to glucose-6-phosphatase (G6Pase) deficiency. An adeno-associated virus (AAV) containing a small human G6Pase transgene was pseudotyped with AAV8 (AAV2/8) to optimize liver tropism. Survival was prolonged in 2-week-old G6Pase (-/-) mice by 600-fold fewer AAV2/8 vector particles (vp), in comparison to previous experiments involving this model (2 x 10(9) vp; 3 x 10(11) vp/kg). When the vector was pseudotyped with AAV1, survival was prolonged only at a higher dose (3 x 10(13) vp/kg). The AAV2/8 vector uniquely prevented hypoglycemia during fasting and fully corrected liver G6Pase deficiency in GSD-Ia mice and dogs. The AAV2/8 vector has prolonged survival in three GSD-Ia dogs to >11 months, which validated this strategy in the large animal model for GSD-Ia. Urinary biomarkers, including lactate and 3-hydroxybutyrate, were corrected by G6Pase expression solely in the liver. Glycogen accumulation in the liver was reduced almost to the normal level in vector-treated GSD-Ia mice and dogs, as was the hepatocyte growth factor (HGF) in GSD-Ia mice. These preclinical data demonstrated the efficacy of correcting hepatic G6Pase deficiency, and support the further preclinical development of AAV vector-mediated gene therapy for GSD-Ia.