Viral Vectors for Gene Therapy: Translational and Clinical Outlook.

In a range of human trials, viral vectors have emerged as safe and effective delivery vehicles for clinical gene therapy, particularly for monogenic recessive disorders, but there has also been early work on some idiopathic diseases. These successes have been enabled by research and development efforts focusing on vectors that combine low genotoxicity and immunogenicity with highly efficient delivery, including vehicles based on adeno-associated virus and lentivirus, which are increasingly enabling clinical success. However, numerous delivery challenges must be overcome to extend this success to many diseases; these challenges include developing techniques to evade preexisting immunity, to ensure more efficient transduction of therapeutically relevant cell types, to target delivery, and to ensure genomic maintenance. Fortunately, vector-engineering efforts are demonstrating promise in the development of next-generation gene therapy vectors that can overcome these barriers. This review highlights key historical trends in clinical gene therapy, the recent clinical successes of viral-based gene therapy, and current research that may enable future clinical application.

Gene therapy with recombinant adeno-associated vectors for neovascular age-related macular degeneration: 1 year follow-up of a phase 1 randomised clinical trial.

BACKGROUND: Neovascular, or wet, age-related macular degeneration causes central vision loss and represents a major health problem in elderly people, and is currently treated with frequent intraocular injections of anti-VEGF protein. Gene therapy might enable long-term anti-VEGF therapy from a single treatment. We tested the safety of rAAV.sFLT-1 in treatment of wet age-related macular degeneration with a single subretinal injection. METHODS: In this single-centre, phase 1, randomised controlled trial, we enrolled patients with wet age-related macular degeneration at the Lions Eye Institute and the Sir Charles Gairdner Hospital (Nedlands, WA, Australia). Eligible patients had to be aged 65 years or older, have age-related macular degeneration secondary to active subfoveal choroidal neovascularisation, with best corrected visual acuity (BCVA) of 3/60-6/24 and 6/60 or better in the other eye. Patients were randomly assigned (3:1) to receive either 1 × 10(10) vector genomes (vg; low-dose rAAV.sFLT-1 group) or 1 × 10(11) vg (high-dose rAAV.sFLT-1 group), or no gene-therapy treatment (control group). Randomisation was done by sequential group assignment. All patients and investigators were unmasked. Staff doing the assessments were masked to the study group at study visits. All patients received ranibizumab at baseline and week 4, and rescue treatment during follow-up based on prespecified criteria including BCVA measured on the Early Treatment Diabetic Retinopathy Study (EDTRS) scale, optical coherence tomography, and fluorescein angiography. The primary endpoint was ocular and systemic safety. This trial is registered with ClinicalTrials.gov, number NCT01494805. FINDINGS: From Dec 16, 2011, to April 5, 2012, we enrolled nine patients of whom eight were randomly assigned to receive either intervention (three patients in the low-dose rAAV.sFLT-1 group and three patients in the high-dose rAAV.sFLT-1 group) or no treatment (two patients in the control group). Subretinal injection of rAAV.sFLT-1 was highly reproducible. No drug-related adverse events were noted; procedure-related adverse events (subconjunctival or subretinal haemorrhage and mild cell debris in the anterior vitreous) were generally mild and self-resolving. There was no evidence of chorioretinal atrophy. Clinical laboratory assessments generally remained unchanged from baseline. Four (67%) of six patients in the treatment group required zero rescue injections, and the other two (33%) required only one rescue injection each. INTERPRETATION: rAAV.sFLT-1 was safe and well tolerated. These results support ocular gene therapy as a potential long-term treatment option for wet age-related macular degeneration. FUNDING: National Health and Medical Research Council of Australia, Richard Pearce Bequest, Lions Save Sight Foundation, Brian King Fellowship, and Avalanche Biotechnologies, Inc.

Modulation of transgene expression in retinal gene therapy by selective laser treatment.

PURPOSE: To develop a method for modulation of transgene expression in retinal pigment epithelium (RPE) using scanning laser that spares neurosensory retina. METHODS: Fifteen pigmented rabbits received subretinal injection of recombinant adeno-associated virus (rAAV-2) encoding green fluorescent protein (GFP). GFP expression was measured using confocal scanning laser ophthalmoscopy (cSLO) fluorescence imaging and immunohistochemistry. To reduce the total expression in RPE by half, 50% of the transfected RPE cells were selectively destroyed by microsecond exposures to scanning laser with 50% pattern density. The selectivity of RPE destruction and its migration and proliferation were monitored using fluorescein angiography, spectral-domain optical coherence tomography (SD-OCT), and light, transmission, and scanning electron microscopy. 5-Bromo-2'-dioxyuridine (BrdU) assay was performed to evaluate proliferation of RPE cells. RESULTS: RPE cells were selectively destroyed by the line scanning laser with 15 µs exposures, without damage to the photoreceptors or Bruch's membrane. RPE cells started migrating after the first day, and in 1 week there was complete restoration of RPE monolayer. Selective laser treatment decreased the GFP fluorescence by 54% as compared to control areas; this was further decreased by an additional 48% following a second treatment 1 month later. BrdU assay demonstrated proliferation in approximately half of the RPE cells in treatment areas. CONCLUSIONS: Microsecond exposures produced by scanning laser destroyed RPE cells selectively, without damage to neural retina. Continuity of RPE layer is restored within days by migration and proliferation, but transgene not integrated into the nucleus is not replicated. Therefore, gene expression can be modulated in a precise manner by controlling the laser pattern density and further adjusted using repeated applications.

Integration specificity of phage phiC31 integrase in the human genome.

The site-specific integrase from bacteriophage phiC31 functions in mammalian cells and is being applied for genetic engineering, including gene therapy. The phiC31 integrase catalyzes precise, unidirectional recombination between its 30-40-bp attP and attB recognition sites. In mammalian cells, the enzyme also mediates integration of plasmids bearing attB into native sequences that have partial sequence identity with attP, termed pseudo attP sites. Here, we analyzed the features of phiC31-mediated integration into pseudo attP sites in the human genome. Sequence analysis of 196 independent integration events derived from three cell lines revealed approximately 101 integration sites: 56% of the events were recurrent integrations distributed among 19 pseudo attP sequences. Bioinformatics analysis revealed a approximately 30-bp palindromic consensus sequence motif shared by all of the repeat occurrences and most of the single occurrence sites, verifying that phiC31-mediated integration into pseudo attP sites is significantly guided by DNA sequence recognition. The most favored unique sequence in these cell lines occurred at chromosome 19q13.31 and accounted for 7.5% of integration events. Other frequent integration sites were in three specific sequences in subfamilies of ERVL and L1 repetitive sequences, accounting for an additional 17.9% of integration events. Integrations could occur in either orientation at a pseudo attP site, were often accompanied by small deletions, and typically occurred in a single copy per cell. A number of aberrant events were also described, including large deletions and chromosome rearrangements. phiC31 integrase-mediated integration only slightly favored genes and did not favor promoter regions. Gene density and expression studies suggested chromatin context effects. An analysis of the safety of integration sites in terms of proximity to cancer genes suggested minimal cancer risk. We conclude that integration systems derived from phiC31 integrase have great potential utility.