Thomas W. Chalberg

Site-specific genomic integration produces therapeutic Factor IX levels in mice

We used the integrase from phage φC31 to integrate the human Factor IX (hFIX) gene permanently into specific sites in the mouse genome. A plasmid containing attB and an expression cassette for hFIX was delivered to the livers of mice by using high-pressure tail vein injection. When an integrase expression plasmid was co-injected, hFIX serum levels increased more than tenfold to ∼4 μg/ml, similar to normal FIX levels, and remained stable throughout the more than eight months of the experiment. hFIX levels persisted after partial hepatectomy, suggesting genomic integration of the vector. Site-specific integration was proven by characterizing and quantifying genomic integration in the liver at the DNA level. Integration was documented at two pseudo-attP sites, native sequences with partial identity to attP, with one site highly predominant. This study demonstrates in vivo gene transfer in an animal by site-specific genomic integration.

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.

Plasma-Mediated Transfection of RPE

A major obstacle in applying gene therapy to clinical practice is the lack of efficient and safe gene delivery techniques. Viral delivery has encountered a number of serious problems including immunological reactions and malignancy. Non-viral delivery methods (liposomes, sonoporation and electroporation) have either low efficiency in-vivo or produce severe collateral damage to ocular tissues. We discovered that tensile stress greatly increases the susceptibility of cellular membranes to electroporation. For synchronous application of electric field and mechanical stress, both are generated by the electric discharge itself. A pressure wave is produced by rapid vaporization of the medium. To prevent termination of electric current by the vapor cavity it is ionized thus restoring its electric conductivity. For in-vivo experiments with rabbits a plasmid DNA was injected into the subretinal space, and RPE was treated trans-sclerally with an array of microelectodes placed outside the eye. Application of 250-300V and 100-200 μs biphasic pulses via a microelectrode array resulted in efficient transfection of RPE without visible damage to the retina. Gene expression was quantified and monitored using bioluminescence (luciferase) and fluorescence (GFP) imaging. Transfection efficiency of RPE with this new technique exceeded that of standard electroporation by a factor 10,000. Safe and effective non-viral DNA delivery to the mammalian retina may help to materialize the enormous potential of the ocular gene therapy. Future experiments will focus on continued characterization of the safety and efficacy of this method and evaluation of long-term transgene expression in the presence of phiC31 integrase.

394. Baseline Data for Patients Participating in the Phase 2a rAAV.sFlt-1 Gene Therapy Trial for Exudative Age-Related Macular Degeneration

rAAV.sFlt-1 has the potential to provide long-term treatment of retinal neovascular diseases following a single injection by driving expression of sFlt-1, a potent (Kd 10 pM), naturally-occurring VEGF inhibitor. Here we present the baseline data for subjects with exudative age-related macular degeneration (AMD) enrolled in an investigator-sponsored, single center, Phase 2a safety study of rAAV.sFlt-1 gene therapy. Of the 32 randomized subjects, 21 were randomized to the rAAV.sFlt-1 group and 11 to the ranibizumab control group. All subjects were treated with two initial intravitreal injections of ranibizumab and then followed monthly for the need for rescue ranibizumab injections based on pre-specified criteria for evidence of active exudative AMD. Subjects underwent a thorough ophthalmic examination at baseline, and clinical laboratory assessments included blood biochemistry, complete blood count, and T-cell response. Anti-AAV antibodies (both total and neutralizing), AAV capsid protein, and sFlt-1 protein levels were also measured. Baseline demographics were 100% Caucasian, 59% female, and average age 79+7 years. All subjects had active subfoveal choroidal neovascularization secondary to exudative AMD, and the average visual acuity was 59+15 EDTRS letters (Snellen equivalent 20/63). Subjects had received an average of 10 anti-VEGF injections prior to enrolment. Anti-AAV antibody results indicated that 25% of subjects serum contained neutralizing antibody titres 1/100-1/500 while 19% had titres 1/20-1/100. All subjects tested negative for presence of AAV capsid in fluids at baseline. sFlt-1 protein averaged 13.9+5.4 pg/ml in urine, 116+5.9 pg/ml in serum, and 229+12.7 pg/ml in saliva. One patient had IgM deficiency and 6/32 patients had lymphopenia or T cell deficiency and/or CD8 lymphopenia. No other immune defects that might pose a hazard to the use of the rAAV vector were identified. In conclusion, the study subjects had characteristics representative of a population with exudative AMD and are appropriate for enrollment in a safety study for rAAV.sFlt-1.

202. pMNTC Is a Cone-Specific Regulatory Cassette Designed To Treat Cone-Associated Disorders

New therapies are needed for the treatment of many cone photoreceptor associated disorders, including macular dystrophies such as cone-rod dystrophy, progressive cone dystrophy, Stargardt macular dystrophy, and achromatopsia. As these vision disorders are associated with a loss of function and/or viability of the cone photoreceptors, it is hypothesized that these disorders may be treatable by delivering a therapeutic gene to cone photoreceptors to rescue cone viability and function. To optimize efficient expression in cone cells, the regulatory cassette “pMNTC” was designed in which enhancer, promoter, 5’UTR, intron, Kozak, and polyadenylation sequences were optimized for cone-specific expression. pMNTC-directed transgene expression was evaluated in a number of species with varying numbers of retinal cones cells among total photoreceptors, including mouse (3% cones), rat (1% cones), gerbil (13% cones), and nonhuman primate (5% cones). pR2.1, a well-characterized cone-specific promoter, was used as a control. AAV vectors carrying a reporter gene under control of the pMNTC regulatory cassette were injected intravitreally. Robust reporter gene expression was seen in the rat, gerbil, and nonhuman primate retina, with expression levels and anatomic location correlating with cone abundance and location in these species. Analysis of transduced mouse retina by immunohistochemistry demonstrated that this expression is highly cone-specific. Abundant expression was seen in nonhuman primate around the fovea. Reporter expression was detected sooner and in more cones in nonhuman primate transduced with the pMNTC expression cassette as compared to the pR2.1 expression cassette. In conclusion, the pMNTC regulatory cassette is an improved regulatory element that may facilitate the development of therapeutic agents for cone-associated disorders.

316. Evaluating AAV Hybrid Variants for Improved Tropism after Intravitreal Gene Delivery to the Retina

AAV-mediated gene therapy has shown promise for some ocular diseases when injected sub-retinally. Intravitreal AAV administration, while less invasive, is far less efficient due to the inner limiting membrane (ILM), which is a particularly profound barrier in the primate retina. Recently AAV2.7m8, which has a 10 amino acid (aa) peptide insertion in the surface exposed loop region of AAV2, was identified using in vivo directed evolution (Dalkara et al., 2013). This variant has been shown to effectively transduce outer retinal cells resulting in extensive gene expression in the non-human primate retina following intravitreal injection.The purpose of our study was to identify AAV variants that could not only penetrate the outer retina from the vitreous, but also have a favorable neutralizing antibody (nAb) titer to allow beneficial AAV-mediated gene expression. We tested whether the 10-aa peptide loop from AAV2.7m8 could be inserted into other capsid backbones, and investigated the tropism and nAb profile of these hybrid capsids. The 10-aa loop was inserted at seven different positions in the receptor binding region of loop IV of capsid 2.5T, which is a chimeric variant of the VP1 region of AAV2 and VP2 and VP3 regions of AAV5 containing a single A581T point mutation (Excoffon et al., 2009). A majority of the 2.5T/7m8 hybrid capsids were successfully packaged and resulted in varying levels of GFP transgene expression following transduction in vitro. Additionally, in vitro transduction was blocked to a lesser extent by nAbs (IVIg) when compared to AAV2, which indicates that the 2.5T/7m8 hybrids may not be impeded by preexisting immunity in human patients. Finally, significantly higher GFP expression was observed with one of the 2.5T/7m8 hybrid variants as compared to the parental 2.5T capsid, following intravitreal administration in rodents.

Optical modulation of transgene expression in retinal pigment epithelium

Over a million people in US alone are visually impaired due to the neovascular form of age-related macular degeneration (AMD). The current treatment is monthly intravitreal injections of a protein which inhibits Vascular Endothelial Growth Factor, thereby slowing progression of the disease. The immense financial and logistical burden of millions of intravitreal injections signifies an urgent need to develop more long-lasting and cost-effective treatments for this and other retinal diseases. Viral transfection of ocular cells allows creation of a “biofactory” that secretes therapeutic proteins. This technique has been proven successful in non-human primates, and is now being evaluated in clinical trials for wet AMD. However, there is a critical need to down-regulate gene expression in the case of total resolution of retinal condition, or if patient has adverse reaction to the trans-gene products. The site for genetic therapy of AMD and many other retinal diseases is the retinal pigment epithelium (RPE). We developed and tested in pigmented rabbits, an optical method to down-regulate transgene expression in RPE following vector delivery, without retinal damage. Microsecond exposures produced by a rapidly scanning laser vaporize melanosomes and destroy a predetermined fraction of the RPE cells selectively. RPE continuity is restored within days by migration and proliferation of adjacent RPE, but since the transgene is not integrated into the nucleus it is not replicated. Thus, the decrease in transgene expression can be precisely determined by the laser pattern density and further reduced by repeated treatment without affecting retinal structure and function.