Hu Sheng Qian

Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle

Although the transient nature of transgene expression using first‐generation adenovirus (Ad) vectors is well known, the exact mechanisms responsible for this phenomenon are uncertain.

Effect of viral dose on neutralizing antibody response and transgene expression after AAV1 vector re-administration in mice

Neutralizing antibodies (nAB) at the time of administration hamper the effectiveness of adeno-associated virus (AAV) as a clinical DNA delivery system. The present study was designed to investigate if AAV re-administration in muscle tissue is dependent on the nAB titer. Recombinant (r)AAV serotype 1, as a promising candidate for targeting skeletal muscle, was used for gene delivery. C57Bl/6 mice were infected intramuscularly with doses between 1 × 109 and 5 × 1010 virus particles (vp) of AAV1-expressing luciferase (AAV1-luc) or human interferon-β (AAV1-hIFNβ). Increasing transgene expression was observed over the first 2 months and anti-AAV1 nAB titers peaked between weeks 4 and 8. Six months after the first administration, 5 × 1010 vp of AAV1-IFNβ were re-administered. Following re-administration, nAB titers increased but did not significantly affect transgene expression from the AAV vector that had been administered first. In contrast, hIFNβ expression originating from the second vector administration was significantly diminished and reflected the nAB titer present at the day of re-administration. The present study extends earlier observations that preexisting nAB affects AAV1 re-administration. The level of nAB is proportional to the virus dose used for the first injection and transgene expression following re-administration is dependent on preexisting nAB titer.

Overexpression of PAI-1 prevents the development of abdominal aortic aneurysm in mice.

Vessel wall inflammation and matrix destruction are critical to abdominal aortic aneurysm (AAA) formation and rupture. We have previously shown that urokinase plasminogen activator (uPA) is highly expressed in experimental AAA and is essential for AAA formation and expansion. In this study, we examined the effects of overexpression of a natural inhibitor of uPA, plasminogen activator inhibitor-1 (PAI-1), on the development of angiotensin (Ang) II-induced AAA in ApoE-deficient (ApoE−/−) mice. Mice were treated with recombinant adenovirus containing either the human PAI-1 gene (Ad5.CMV.PAI-1) or the luciferase gene (Ad5.CMV.Luc) delivered either locally by intra-adventitial injection or systemically by tail vein injection. Our results show that local delivery of the PAI-1 gene completely prevented AAA formation (0 vs 55.6% in Ad5.CMV.Luc controls, P<0.05). In contrast, systemic delivery of the PAI-1 gene did not affect AAA incidence (78 vs 90% in Ad5.CMV.Luc controls, P=0.125). Local delivery of the PAI-1 gene 2 weeks after Ang II infusion prevented further expansion of small aneurysms, but had no significant effect on the progression of larger aneurysms. These data suggest that local PAI-1 gene transfer could be used to stabilize small AAA and reduce the rate of expansion and risk of rupture.

Age-dependent acceleration of ischemic injury in endothelial nitric oxide synthase-deficient mice: potential role of impaired VEGF receptor 2 expression.

Morbidity and mortality of peripheral arterial occlusive disease significantly increases with age, often exhibiting more severe disease pathology and decreased treatment effectiveness. Therapeutic angiogenesis with angiogenic growth factors may represent a valuable treatment option for the severely ill, older adult patient population. Aging is considered an independent cardiovascular risk factor, but pathomechanistically it is not well understood. Diminished endothelial nitric oxide (EDNO) production has been considered as a major contributor to the aging process. To investigate the effect of age on postischemic revascularization independent of changes in EDNO, we used endothelial nitric oxide synthase–deficient (ecNOS-KO) mice. We found an age-dependent acceleration in ischemic injury following unilateral femoral artery ligation in these animals compared to C57BL/J6 mice. Postischemic revascularization, quantified by measuring von Willebrand factor expression, was significantly impaired, suggesting that factors other than progressive EDNO deterioration are also involved in the age-dependent severe disease phenotype. Ischemia led to an increase in the expression of vascular endothelial growth factor receptor-2, KDR, in younger ecNOS-KO; however, this increase in KDR expression was absent in the older animals. Lack of increased KDR expression may provide a mechanistic explanation for the severe ischemic injury and perhaps can be used as a clinical marker to identify severe, vascular endothelial growth factor refractory patient population.

Effective treatment of vascular endothelial growth factor refractory hindlimb ischemia by a mutant endothelial nitric oxide synthase gene

Gene delivery of angiogenic growth factors is a promising approach for the treatment of ischemic cardiovascular diseases. However, success of this new therapeutic principle is hindered by the lack of critical understanding as to how disease pathology affects the efficiency of gene delivery and/or the downstream signaling pathways of angiogenesis. Critical limb ischemia occurs in patients with advanced atherosclerosis often exhibiting deficiency in endothelial nitric oxide production. Similar to these patients, segmental femoral artery resection progresses into severe ischemic necrosis in mice deficient in endothelial nitric oxide synthase (ecNOS-KO) as well as in balb/c mice. We used these models to evaluate the influence of severe ischemia on transfection efficiency and duration of transgene expression in the skeletal muscle following plasmid injection in combination with electroporation. Subsequently, we also explored the potential therapeutic effect of the phosphomimetic mutant of ecNOS gene (NOS1177D) using optimized delivery parameters, and found significant benefit both in ecNOS-KO and balb/c mice. Our results indicate that NOS1177D gene delivery to the ischemic skeletal muscle can be efficient to reverse critical limb ischemia in pathological settings, which are refractory to treatments with a single growth factor, such as vascular endothelial growth factor.

Regulated expression of the interferon-β gene in mice

A single plasmid regulated expression vector based upon a mifepristone-inducible two plasmid system, termed pBRES, has been constructed and tested in mice using murine interferon-b (mIFNb) as the transgene. The expression of mIFNb in the circulation was followed by measuring the systemic induction of IP-10, a validated biomarker for mIFNb in mice. Long-term, inducible expression of mIFNb was demonstrated following a single intramuscular (i.m.) injection of the pBRES mIFNb plasmid vector into the hind limb of mice. Induction of mIFNb expression was achieved by administration of the small molecule inducer, mifepristone (MFP). Plasmid DNA and mIFNb mRNA levels in the injected muscles correlated with mIFNb expression as monitored by IP-10 over a 3-month time period. Renewable transgene expression was achieved following repeat administration of the plasmid at 3 months following the first plasmid injection. A dose-dependent increase in expression was demonstrated by varying the amount of injected plasmid or the amount of the inducer administered to the mice. Finally, the pBRES plasmid expressing mIFNb under control of the inducer, MFP, was shown to be efficacious in a murine model of experimental allergic encephalomyelitis, supporting the feasibility of gene-based therapeutic approaches for treating diseases such as multiple sclerosis.

Role of Endothelial Nitric Oxide in Bone Marrow-Derived Progenitor Cell Mobilization

Mobilization and recruitment of bone marrow-derived progenitor cells (BMDPCs) play an important role in postischemic tissue repair. Patients with coronary artery disease (CAD) or peripheral vascular disease (PVD) exhibit endothelial dysfunction, and as a result are likely to have a reduced number of progenitor cells mobilized in their peripheral circulation following ischemic injury. Identification of eNOS independent pathways for BMDPC mobilization may have important therapeutic value in this patient population. To identify such mechanisms we investigated the effect of granulocyte-colony stimulating factor (GCSF) and stem cell factor (SCF) in eNOS-KO mice with and without surgical hind-limb ischemia. Our results suggest that BMDPC mobilization can be achieved via activation of NO-independent pathways.

768. Two Distinct Mechanisms, Silencing of RNA Expression and Loss of Vector DNA, Are Responsible for the Loss of Transgene Expression Following Delivery of Foreign Genes to Skeletal Muscle

Plasmid based delivery of foreign transgenes often results in transient expression in vivo. To investigate the cause of this transient expression we used quantitative PCR to measure the copy number of transgene RNA and vector DNA in the skeletal muscle of C57BL6 mice after electroporation of various plasmids. Gene transfer of a plasmid encoding the mouse interferon beta (mIFN-|[beta]|) gene driven by the CMV promoter (pCMV-mIFN) resulted in mIFN RNA levels between 1 and 5 |[times]| 106 copies per microgram (|[mu]|g) of total RNA that were stable for the first 10 days then dropped by only 10-fold by day 44. Plasmid vector DNA levels were also stable for the first 10 days then dropped 7.5-fold between day 10 and day 44. The ratio of mIFN RNA copies per vector DNA copy reached a peak of 1.8 at day 10 and declined slightly to 0.9 at day 44. These results show that following delivery of a non-foreign transgene, a slow decline in RNA levels occurred that was due to a gradual loss of plasmid DNA. Next, we compared plasmids encoding either the mIFN-|[beta]| or human interferon beta (hIFN-|[beta]|) genes driven by identical CMV promoters (pCMV-hIFN and pCMV-mIFN). At 50 days after electroporation of mouse skeletal muscle, the mean copy number of transgene RNA in the muscles that received the mouse or human IFN-|[beta]| plasmids was 1.1 |[times]| 105 and 7.4 |[times]| 103 copies per |[mu]|g of total RNA, respectively. In contrast, the mean plasmid DNA level was slightly higher in the muscles that received pCMV-hIFN. The mean IFN-|[beta]| RNA to vector DNA ratio was 1.3 in the muscles that received pCMV-mIFN, but only 0.04 (31-fold lower, p<0.001) in the muscles that received pCMV-hIFN. This demonstrates that the lower level of transgene RNA expression achieved with the foreign human IFN-|[beta]| gene as compared to the native mouse IFN-|[beta]| gene was due to reduced gene expression, not to reduced persistence of the vector DNA. When a mixture of pCMV-mIFN and a plasmid encoding human secreted alkaline phosphotase driven by the CMV promoter (pCMV-hSEAP) in a 14:1 ratio was delivered to mouse muscle, mIFN RNA levels fell 350-fold between day 1 and day 10 (p<0.01). The level of pCMV-mIFN plasmid DNA in the same muscles fell 100-fold by day 10 (p<0.01) and the ratio of mIFN RNA copies per copy of pCMV-mIFN DNA dropped slightly from 2.8 at day 1 to 1.1 at day 10, suggesting that there was only a small decrease in transcription. This data demonstrates that following co-delivery of pCMV-hSEAP and pCMV-mIFN, rapid clearance of the CMV-mIFN plasmid DNA from the muscle was primarily responsible for the large drop in mIFN RNA expression.

959. Pharmacokinetic Studies of Human IFN-|[szlig]| in Mice Following Gene-Based Delivery

Interferon-|[szlig]| (IFN-|[szlig]|) is an immunoregulatory cytokine that has been approved as a protein therapeutic for multiple sclerosis (MS). Pharmacokinetic studies have shown that IFN-|[szlig]| has an extremely short half-life in the circulation. Within a few hours following bolus delivery of the recombinant protein, hIFN-|[szlig]| levels in serum are undetectable. Gene-based delivery of IFN-|[szlig]| would offer advantages over bolus delivery of protein in providing sustained long-term expression of the protein resulting in therapeutic efficacy while minimizing side effects.