Adenoviral vectors for liver-directed gene therapy

Adenoviral vectors currently represent one of the most efficient means of in vivo hepatocyte gene delivery. Consequently, liver-directed gene transfer has been increasingly explored as a promising approach for the treatment of a diverse range of genetic and acquired diseases. Numerous demonstrations of efficacious adenoviral vector-mediated delivery of a wide array of transgenes in several animal species and humans have been reported. In general, transgene expression was efficient, but transient, in many cases lasting < 1 month. Currently, efforts in the field are focused on the development of highly attenuated adenoviral vectors designed to prolong transgene expression by reducing vector immunogenicity and hepatoxicity. Vector optimization strategies include the development of vectors devoid of all viral coding regions, the generation of chimeric vectors engineered to capitalize on favorable aspects of the component viral systems, the development of tissue-specific regulated gene expression, and the development of strategies to circumvent the host immune system. The use of adenoviral vectors for gene therapy of hereditary, malignant and infectious diseases of the liver, and the vector optimization strategies outlined above are discussed in this work.     

Hepatic imaging following intra-arterial embolotherapy

Purpose

To discuss guidelines and salient imaging findings of solid tumors treated with common intra-arterial procedures used in interventional oncology.

Methods

A meticulous literature search of PubMed-indexed articles was conducted. Key words included “imaging + embolization,” “imaging + TACE,” “imaging + radioembolization,” “imaging + Y90,” “mRECIST,” and “EASL.” Representative post-treatment cross-sectional images were obtained from past cases in this institution.

Results

Intra-arterial therapy (IAT) in interventional oncology includes bland embolization, chemoembolization, and radioembolization. Solid tumors of the liver are the primary focus of these procedures. Cross-sectional CT and/or MR are the main modalities used to image tumors after treatment. Traditional size-based response criteria (WHO and RECIST) alone are of limited utility in determining response to IAT; tumoral necrosis and enhancement must be considered. Specifically for HCC, the EASL and mRECIST guidelines are becoming widely adopted response criteria to assess these factors. DWI, FDG-PET, and CEUS are modalities that play an adjunctive but controversial role.

Conclusions

Radiologists must be aware that the different forms of intra-arterial therapy yield characteristic findings on cross-sectional imaging. Knowledge of these findings is integral to accurate assessment of tumor response and progression.

     

Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria

Novel recombinant adeno-associated virus vectors pseudotyped with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pah(enu2) mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pah(enu2) mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.5+/-2.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism.     

儿童恶性肿瘤放射治疗并发症的研究进展

儿童恶性肿瘤治疗后的长期生存者是各医疗保健专家公认的患病高风险人口,对肿瘤的各种治疗手段都会不同程度的影响到儿童的生长和发育。放疗及化疗后的幸存者,将经历慢性或迟发性身体健康问题。成长中的儿童受放疗影响尤其敏感,受照野内的器官和组织、放射源的选择、分割方式、累积照射剂量和不同的受照年龄都会对放疗反应的轻重有明显的影响。     

Elderly patients and intra-arterial stroke therapy

Ischemic stroke disproportionately affects the elderly, particularly those over the age of 80 years. Rates of stroke are expected to increase over the next several decades due to increasing numbers of elderly individuals, making understanding stroke treatment in this population an imperative. The only proven acute stroke therapy is early reperfusion, accomplished through intravenous or intra-arterial means. Intra-arterial stroke therapy (IAT) offers higher recanalization rates than intravenous tissue plasminogen activator, but has yet to show clear superiority over intravenous tissue plasminogen activator alone. Existing data suggest that elderly stroke patients suffer worse outcomes following IAT, despite similar rates of recanalization and symptomatic intracranial hemorrhage. This article reviews the application of IAT in the elderly population and summarizes the available studies that investigate the response of elderly patients to IAT.     

Limited transgene immune response and long-term expression of human alpha-L-iduronidase in young adult mice with mucopolysaccharidosis type I by liver-directed gene therapy

Mucopolysaccharidosis type I (MPS I) due to deficient alpha-L-iduronidase (IDUA) activity results in the accumulation of glycosaminoglycans (GAGs) in many of the cells of affected patients. Stable gene replacement by in vivo administration of lentiviral vectors (LVs) has therapeutic potential for metabolic disorders and other systemic diseases. We have previously shown in a murine model the therapeutic potential of lentiviral IDUA vector-mediated gene therapy, in which human IDUA cDNA was driven by the cytomegalovirus promoter. However, the major limitation of this approach was the induction of an immune response against the therapeutic protein, which limited the efficacy and long-term duration of treatment. In this study, we evaluate the potential of liver-directed gene therapy, that is, programming of murine hepatocytes to secrete the enzyme with mannose 6-phosphate (M6P), which can be taken up by distant cells. Eight- to 10-week-old mice were injected via the tail vein with a lentiviral vector expressing human IDUA cDNA driven by the albumin gene promoter selectively expressed in hepatocytes. One month after treatment, IDUA activity was present in the liver and spleen of treated mice; an expression level of 1% normal IDUA activity was sufficient to reduce the GAG level in liver, spleen, kidney, heart, and lung. Interestingly, 6 months after a single injection of this vector, IDUA activity was detectable in several murine tissues; the level of enzyme activity was low but sufficient to maintain the decrease in GAG levels in liver, spleen, kidney, heart, and lung. Also, the level of enzyme-specific antibodies reached at 6 months postinjection was nearly null, and real-time polymerase chain reaction analysis showed high levels of vector DNA content in liver and spleen. Thus, these results show that the use of LV with the albumin gene promoter selectively expressed in hepatocytes limited the immune response to the transgene and allowed stable and prolonged expression of the IDUA enzyme and a partial correction of the pathology.     

Imaging tumor angiogenesis

Since the discovery of vascular-specific growth factors with angiogenic activity, there has been a significant effort to develop cancer drugs that restrict tumorigenesis by targeting the blood supply. In this issue of the JCI, Mancuso et al. use mouse models to better understand the plasticity of the tumor vasculature in the face of antiangiogenic therapy (see the related article beginning on page 2610). They describe a rapid regrowth of the tumor vasculature following withdrawal of VEGFR inhibitors, emphasizing the importance of fully understanding the function of these and similar treatments used in the clinic at the cellular and molecular level.     

多西紫杉醇经动脉或静脉灌注对后肢VX2瘤兔的影响

目的 探讨多西紫杉醇经动脉灌注治疗肿瘤的有效性及安全性.方法 建立兔后肢VX2瘤动物模型.经股动脉或耳像静脉灌注多西紫杉醇.比较两种给药方式下实验兔的肿瘤组织、胃壁、肝脏、肾脏、血浆的药物浓度差异.采用t检验作统计学分析.结果 经动脉灌注组的肿瘤组织、胃壁、肝脏及肾脏的药物浓度均高于经静脉灌注组.肿瘤组织及胃壁中的药物浓度差异有统计学意义（P〈0.05）.肿瘤组织的药物浓度差别约为14倍.经动脉灌注组的血浆药物浓度低于经静脉灌注组,两者差异没有统计学意义（P〉0.05）.结论 多西紫杉醇经动脉灌注可提高肿瘤的局部控制效果,但有可能增加毒副反应发生的风险,应慎重采用或减少用药剂量.     

Gastroparesis following traumatic brain injury and response to metoclopramide therapy

Most studies on nutrition in patients with traumatic brain injury (TBI) have focused on the hypermetabolic state immediately after trauma, or the swallowing disorders that frequently follow acute TBI. A less well-known sequela, however, is gastroparesis, which generally manifests itself when patients have advanced from nasogastric to oral feeding programs. Case histories of four patients are reported, all of whom responded to therapeutic intervention with metoclopramide. Results of a radionuclide gastric emptying study, before and after metoclopramide therapy, are presented. The etiologic and physiologic mechanisms behind this disorder are unclear, but demonstration of this disorder and other clinical trials with metoclopramide appear to deserve further consideration in the nutritionally compromised TBI patient.     

Acute ischemia of the hand following intra-arterial oxymetazoline injection

Acute ischemia of the hand following intraarterial injection of a drug is an infrequent problem, but it requires urgent medical attention. In this report, a patient injected a nasal decongestant, oxymetazoline hydrochloride (Afrin^®), into his radial artery. This resulted in severe burning pain, and a cyanotic, cold, and pulseless hand, suggestive of acute ischemia. Intra-arterial papaverine, nifedipine, anticoagulation with heparin, prednisone, and stellate ganglion block did not improve the hand circulation. Development of gangrene necessitated a disarticulation at the wrist joint. Diagnosis, complications, and therapeutic measures of acute hand ischemia secondary to intra-arterial drug injection are discussed.     

Pseudo-hydrodynamic delivery of helper-dependent adenoviral vectors into non-human primates for liver-directed gene therapy.

Helper-dependent adenoviral vectors (HDAds) are attractive for liver-directed gene therapy because they can mediate long-term, high-level transgene expression without chronic toxicity. However, systemic delivery requires high vector doses for efficient hepatic transduction, resulting in dose-dependent acute toxicity. Clearly, strategies to improve hepatic transduction with low vector doses are needed. In this regard, we have previously shown that hydrodynamic injection of helper-dependent adenoviral vectors into mice results in increased hepatic transduction, reduced systemic vector dissemination, and reduced pro-inflammatory cytokines compared with conventional injection and thus has the potential to improve dramatically the therapeutic index of helper-dependent adenoviral vectors. Unfortunately, the rapid, large-volume injection used in this method cannot be applied to larger animals. Therefore, we have developed a novel balloon occlusion catheter-based method to mimic hydrodynamic injection of helper-dependent adenoviral vectors into non-human primates that does not require rapid, large-volume injection. Using a low, clinically relevant vector dose, this minimally invasive method results in high-efficiency hepatic transduction with minimal toxicity and stable long-term transgene expression for at least 413 days.     

Imaging of multistep human hepatocarcinogenesis by CT during intra-arterial contrast injection

Various types of hepatocellular nodules are seen in cirrhotic livers. In these nodules, two types of human hepatocarcinogenesis are now considered. One is de novo hepatocarcinogenesis and the other is the stepwise development from high-grade dysplastic nodule (DN), high-grade DN with well-differentiated HCC foci, and overt HCC. According to our analysis by CT during arterial portography (CTAP) and CT during hepatic arteriography (CTHA) and histological study, in accordance with the elevation of the grade of malignancy of the nodules, the portal tract including normal portal vein (intranodular portal supply) and hepatic artery (intranodular arterial supply through normal hepatic arteries) are decreased. On the other hand, abnormal artery (intranodular arterial supply through newly formed abnormal arteries) gradually increases. Therefore, we can estimate the grade of malignacy of the nodules from intranodular blood supply. To know this blood supply pattern is important for the early detection, characterization and treatment of early stage HCCs. We also revealed that there was a close correlation between the prognosis of the nodules and the blood supply patterns.     

Humoral immune response following extracorporeal immunoadsorption therapy of patients with hypercholesterolemia

Low-density lipoprotein apheresis (LDL-apheresis) is an extracorporeal procedure that preferentially removes LDL cholesterol from the blood. One of the primary techniques for performing this procedure uses immunoadsorption columns containing monospecific polyclonal sheep antibodies to human LDL covalently coupled to a gel filtration medium. LDL-apheresis has generally been well-tolerated, with chills, fever, or flushing occurring rarely. The possibility of an immune reaction was investigated as a basis for these reactions observed in 12 of the 1312 procedures performed. Antibodies to sheep IgG developed in 12 of the 15 patients treated with LDL-apheresis as a result of the shedding of small quantities of the sheep immunoglobulin from the columns. A column acid-washing procedure minimized the quantity of shed antibody but did not prevent immunization of the patient. The clinical reactions were probably unrelated to shedding and immunization, as the reactions occurred even in patients who were not immunized to the sheep IgG. Immunization to ethylene oxide was not the cause, as determined by a radioallergosorbent test. The reactions were more likely related to the activation of complement, as indicated by the generation of C3a des Arg by the columns and an increase in C3a des Arg levels systemically.     

Notch promotes recurrence of dormant tumor cells following HER2/neu-targeted therapy

Breast cancer mortality is principally due to recurrent tumors that arise from a reservoir of residual tumor cells that survive therapy. Remarkably, breast cancers can recur after extended periods of clinical remission, implying that at least some residual tumor cells pass through a dormant phase prior to relapse. Nevertheless, the mechanisms that contribute to breast cancer recurrence are poorly understood. Using a mouse model of recurrent mammary tumorigenesis in combination with bioinformatics analyses of breast cancer patients, we have identified a role for Notch signaling in mammary tumor dormancy and recurrence. Specifically, we found that Notch signaling is acutely upregulated in tumor cells following HER2/neu pathway inhibition, that Notch signaling remains activated in a subset of dormant residual tumor cells that persist following HER2/neu downregulation, that activation of Notch signaling accelerates tumor recurrence, and that inhibition of Notch signaling by either genetic or pharmacological approaches impairs recurrence in mice. Consistent with these findings, meta-analysis of microarray data from over 4,000 breast cancer patients revealed that elevated Notch pathway activity is independently associated with an increased rate of recurrence. Together, these results implicate Notch signaling in tumor recurrence from dormant residual tumor cells and provide evidence that dormancy is a targetable stage of breast cancer progression.     

Imaging of neuroblastoma and Wilms' tumor

Neuroblastoma and Wilms' tumor are the most common noncentral nervous system solid tumors in children. Imaging plays a crucial role in the evaluation of the primary tumor and regional and metastatic disease. There is a growing body of literature supporting the use of MRI as the technique of choice for the evaluation of local and regional disease in children with suspected neuroblastoma; however, in children with suspected Wilms' tumor, MRI will likely continue to play a role as a problem-solver when the results of CT are equivocal or indeterminant.