The Unusual Properties of the AAV Inverted Terminal Repeat.

Although the sequence of the AAV inverted terminal repeat has been known for 40 years, there are still unanswered questions about functions attributable to the terminal 125 nucleotides.

Next Generation of Adeno-Associated Virus Vectors for Gene Therapy for Human Liver Diseases.

Recombinant vectors based on a nonpathogenic parvovirus, the adeno-associated virus (AAV), have taken center stage in the past decade. The safety of AAV vectors in clinical trials and clinical efficacy in several human diseases are now well documented. Despite these achievements, it is increasingly clear that the full potential of AAV vectors composed of the naturally occurring capsids is unlikely to be realized. This article describes advances that have been made and challenges that remain in the optimal use of AAV vectors in human gene therapy applications.

AAV: An Overview of Unanswered Questions.

AAV has been studied for 55 years and has been developed as a vector for about 35 years. By now, there is a fairly good idea of the dimensions of what would be useful to know to employ AAV optimally as a vector, but there are still many unanswered questions within the system. As with all biological systems, each good experiment raises further questions to answer. This article provides an overview of those areas in which unknown information can be identified and of those questions that have not yet been recognized. Some of these are touched on in the six review articles in this issue of Human Gene Therapy.

The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG).

Recombinant viral vectors provide an effective means for heterologous antigen expression in vivo and thus represent promising platforms for developing novel vaccines against human pathogens from Ebola to tuberculosis. An increasing number of candidate viral vector vaccines are entering human clinical trials. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to improve our ability to anticipate potential safety issues and meaningfully assess or interpret safety data, thereby facilitating greater public acceptance when licensed.

My life with adeno-associated virus: a long time spent studying a short genome.

My 45 years of studying the molecular biology of adeno-associated virus are recounted. Additional activities as a mentor, department chair, and medical school administrator are described, as are my activities in the public sphere, which involved national issues related to science policy and medical education.

Adeno-associated virus site-specific integration is mediated by proteins of the nonhomologous end-joining pathway.

Adeno-associated virus type 2 (AAV 2) is the only eukaryotic virus capable of site-specific integration; the target site is at chromosome 19q13.4, a site termed AAVS1. The biology of AAV latency has been extensively studied in cell culture, yet the precise mechanism and the required cellular factors are not known. In this study, we assessed the relative frequencies of stable site-specific integration by characterization of cell clones containing integrated AAV vectors. By this assay, two proteins involved in nonhomologous end joining (NHEJ), DNAPKcs and ligase IV, exhibit differential effects on AAV site-specific integration. DNAPKcs is not required; its presence increases the frequency of junction formation indicative of site-specific integration, but seems to reduce the ratio of site-specific integration to random integration (i.e., the latter is even more enhanced). In contrast, site-specific integration is significantly reduced relative to random integration in cells deficient in ligase IV expression. Furthermore, we show that single-stranded AAV vectors are better substrates for site-specific integration than are self-complementary AAV vectors; the absence of DNAPKcs did not affect the targeted integration of these double-stranded AAV vectors. Together, these data suggest that NHEJ proteins participate in site-specific integration, and indicate a role for the single-stranded form of AAV DNA in targeted integration.

Gene therapy using adeno-associated virus vectors.

SUMMARY: The unique life cycle of adeno-associated virus (AAV) and its ability to infect both nondividing and dividing cells with persistent expression have made it an attractive vector. An additional attractive feature of the wild-type virus is the lack of apparent pathogenicity. Gene transfer studies using AAV have shown significant progress at the level of animal models; clinical trials have been noteworthy with respect to the safety of AAV vectors. No proven efficacy has been observed, although in some instances, there have been promising observations. In this review, topics in AAV biology are supplemented with a section on AAV clinical trials with emphasis on the need for a deeper understanding of AAV biology and the development of efficient AAV vectors. In addition, several novel approaches and recent findings that promise to expand AAV's utility are discussed, especially in the context of combining gene therapy ex vivo with new advances in stem or progenitor cell biology.

Phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 alphal-antitrypsin (AAT) vector in AAT-deficient adults.

A phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 (rAAV2) alpha1-antitrypsin (AAT) vector was performed in 12 AAT-deficient adults, 10 of whom were male. All subjects were either homozygous for the most common AAT mutation (a missense mutation designated PI*Z) or compound heterozygous for PI*Z and another mutation known to cause disease. There were four dose cohorts, ranging from 2.1 x 10(12) vector genomes (VG) to 6.9 x 10(13) VG, with three subjects per cohort. Subjects were injected sequentially in a dose-escalating fashion with a minimum of 14 days between patients. Subjects who had been receiving AAT protein replacement discontinued that therapy 28 days before vector administration. There were no vector-related serious adverse events in any of the 12 participants. Vector DNA sequences were detected in the blood between 1 and 3 days after injection in nearly all patients receiving doses of 6.9 x 10(12) VG or higher. Anti-AAV2 capsid antibodies were present and rose after vector injection, but no other immune responses were detected. One subject who had not been receiving protein replacement exhibited low-level expression of wild-type M-AAT in the serum (82 nM), which was detectable 30 days after receiving an injection of 2.1 x 10(13) VG. Unfortunately, residual but declining M-AAT levels from the washout of the protein replacement elevated background levels sufficiently to obscure any possible vector expression in that range in most of the other individuals in the higher dose cohorts.

Proceedings of the Third International Symposium on Retinopathy of Prematurity: an update on ROP from the lab to the nursery (November 2003, Anaheim, California).

The Third International Symposium on Retinopathy of Prematurity (ROP) was convened with the aim of cross fertilizing the horizons of basic and clinical scientists with an interest in the pathogenesis and management of infants with ROP. Ten speakers in the clinical sciences and ten speakers in the basic sciences were recruited on the basis of their research to provide state of the art talks. The meeting was held November 9, 2003 immediately prior to the American Academy of Ophthalmology meeting; scholarships were provided for outreach to developing countries and young investigators. This review contain the summaries of the 20 platform presentations prepared by the authors and the abstracts of presented posters. Each author was asked to encapsulate the current state of understanding, identify areas of controversy, and make recommendations for future research. The basic science presentations included insights into the development of the human retinal vasculature, animal models for ROP, growth factors that affect normal development and ROP, and promising new therapeutic approaches to treating ROP like VEGF targeting, inhibition of proteases, stem cells, ribozymes to silence genes, and gene therapy to deliver antiangiogenic agents. The clinical presentations included new insights into oxygen management, updates on the CRYO-ROP and ETROP studies, visual function in childhood following ROP, the neural retina in ROP, screening for ROP, management of stage 3 and 4 ROP, ROP in the third world, and the complications of ROP in adult life. The meeting resulted in a penetrating exchange between clinicians and basic scientists, which provided great insights for conference attendees. The effect of preterm delivery on the normal cross-talk of neuroretinal and retinal vascular development is a fertile ground for discovering new understanding of the processes involved both in normal development and in retinal neovascular disorders. The meeting also suggested promising potential therapeutic interventions on the horizon for ROP.

Adeno-associated virus-mediated expression of vascular endothelial growth factor peptides inhibits retinal neovascularization in a mouse model of oxygen-induced retinopathy.

Vascular endothelial growth factor (VEGF) has been demonstrated to be a key stimulator of retinal neovascularization (NV), the most common cause of severe and progressive vision loss. In this study, we used a mouse model of oxygen-induced retinopathy (OIR) to explore the potential of gene expression and secretion of short VEGF peptides as a treatment. Peptide-encoding fragments of exons 6 and 7 of the VEGF gene were cloned into a recombinant adeno-associated virus (rAAV) vector. Expression of each peptide in vector-injected eyes was confirmed by reverse transcription-polymerase chain reaction and Western blot analysis. Intravitreal injection of each rAAV vector inhibited retinal NV by 71-83% (p < 0.001) compared with contralateral control eyes in the OIR mouse. Injection and expression of these peptides did not seem to affect the normal appearance of the retina. The results demonstrated that exon 6- and 7-derived VEGF peptides effectively inhibited oxygen-induced retinal NV. Therefore, these VEGF peptides have potential in the treatment of angiogenesis-associated retinal diseases in humans.

Interleukin 10 attenuates neointimal proliferation and inflammation in aortic allografts by a heme oxygenase-dependent pathway.

Interleukin 10 (IL-10) is a pleiotropic cytokine with well known antiinflammatory, immunosuppressive, and immunostimulatory properties. Chronic allograft rejection, characterized by vascular neointimal proliferation, is a major cause of organ transplant loss, particularly in heart and kidney transplant recipients. In a Dark Agouti to Lewis rat model of aortic transplantation, we evaluated the effects of a single intramuscular injection of a recombinant adeno-associated viral vector (serotype 1) encoding IL-10 (rAAV1-IL-10) on neointimal proliferation and inflammation. rAAV1-IL-10 treatment resulted in a significant reduction of neointimal proliferation and graft infiltration with macrophages and T and B lymphocytes. The mechanism underlying the protective effects of IL-10 in aortic allografts involved heme oxygenase 1 (HO-1) because inhibition of HO activity reversed not only neointimal proliferation but also inflammatory cell infiltration. Our results indicate that IL-10 attenuates neointimal proliferation and inflammatory infiltration and strongly imply that HO-1 is an important intermediary through which IL-10 regulates the inflammatory responses associated with chronic vascular rejection.