Settimio Rossi

Safety and Efficacy of Gene Transfer for Leber’s Congenital Amaurosis

Lebers congenital amaurosis (LCA) is a group of inherited blinding diseases with onset during childhood. One form of the disease, LCA2, is caused by mutations in the retinal pigment epithelium-specific 65-kDa protein gene (RPE65). We investigated the safety of subretinal delivery of a recombinant adeno-associated virus (AAV) carrying RPE65 complementary DNA (cDNA) (ClinicalTrials.gov number, NCT00516477 [ClinicalTrials.gov]). Three patients with LCA2 had an acceptable local and systemic adverse-event profile after delivery of AAV2.hRPE65v2. Each patient had a modest improvement in measures of retinal function on subjective tests of visual acuity. In one patient, an asymptomatic macular hole developed, and although the occurrence was considered to be an adverse event, the patient had some return of retinal function. Although the follow-up was very short and normal vision was not achieved, this study provides the basis for further gene therapy studies in patients with LCA.

Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial

BACKGROUND Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We therefore did a phase 1 trial to assess the effect of gene therapy on retinal and visual function in children and adults with Lebers congenital amaurosis. METHODS We assessed the retinal and visual function in 12 patients (aged 8-44 years) with RPE65-associated Lebers congenital amaurosis given one subretinal injection of adeno-associated virus (AAV) containing a gene encoding a protein needed for the isomerohydrolase activity of the retinal pigment epithelium (AAV2-hRPE65v2) in the worst eye at low (1.5 x 10(10) vector genomes), medium (4.8 x 10(10) vector genomes), or high dose (1.5 x 10(11) vector genomes) for up to 2 years. FINDINGS AAV2-hRPE65v2 was well tolerated and all patients showed sustained improvement in subjective and objective measurements of vision (ie, dark adaptometry, pupillometry, electroretinography, nystagmus, and ambulatory behaviour). Patients had at least a 2 log unit increase in pupillary light responses, and an 8-year-old child had nearly the same level of light sensitivity as that in age-matched normal-sighted individuals. The greatest improvement was noted in children, all of whom gained ambulatory vision. The study is registered with ClinicalTrials.gov, number NCT00516477. INTERPRETATION The safety, extent, and stability of improvement in vision in all patients support the use of AAV-mediated gene therapy for treatment of inherited retinal diseases, with early intervention resulting in the best potential gain. FUNDING Center for Cellular and Molecular Therapeutics at the Childrens Hospital of Philadelphia, Foundation Fighting Blindness, Telethon, Research to Prevent Blindness, F M Kirby Foundation, Mackall Foundation Trust, Regione Campania Convenzione, European Union, Associazione Italiana Amaurosi Congenita di Leber, Fund for Scientific Research, Fund for Research in Ophthalmology, and National Center for Research Resources.

Gene Therapy for Leber's Congenital Amaurosis is Safe and Effective Through 1.5 Years After Vector Administration

The safety and efficacy of gene therapy for inherited retinal diseases is being tested in humans affected with Lebers congenital amaurosis (LCA), an autosomal recessive blinding disease. Three independent studies have provided evidence that the subretinal administration of adeno-associated viral (AAV) vectors encoding RPE65 in patients affected with LCA2 due to mutations in the RPE65 gene, is safe and, in some cases, results in efficacy. We evaluated the long-term safety and efficacy (global effects on retinal/visual function) resulting from subretinal administration of AAV2-hRPE65v2. Both the safety and the efficacy noted at early timepoints persist through at least 1.5 years after injection in the three LCA2 patients enrolled in the low dose cohort of our trial. A transient rise in neutralizing antibodies to AAV capsid was observed but there was no humoral response to RPE65 protein. The persistence of functional amelioration suggests that AAV-mediated gene transfer to the human retina does not elicit immunological responses which cause significant loss of transduced cells. The persistence of physiologic effect supports the possibility that gene therapy may influence LCA2 disease progression. The safety of the intervention and the stability of the improvement in visual and retinal function in these subjects support the use of AAV-mediated gene augmentation therapy for treatment of inherited retinal diseases.

Three-year follow-up after unilateral subretinal delivery of adeno-associated virus in patients with Leber congenital Amaurosis type 2.

OBJECTIVE The aim of this study was to show the clinical data of long-term (3-year) follow-up of 5 patients affected by Leber congenital amaurosis type 2 (LCA2) treated with a single unilateral injection of adeno-associated virus AAV2-hRPE65v2. DESIGN Clinical trial. PARTICIPANTS Five LCA2 patients with RPE65 gene mutations. METHODS After informed consent and confirmation of trial eligibility criteria, the eye with worse visual function was selected for subretinal delivery of adeno-associated virus (AAV2-hRPE65v2). Subjects were evaluated before and after surgery at designated follow-up visits (1, 2, 3, 14, 30, 60, 90, 180, 270, and 365 days, 1.5 years, and 3 years) by complete ophthalmic examination. Efficacy for each subject was monitored with best-corrected visual acuity, kinetic visual field, nystagmus testing, and pupillary light reflex. MAIN OUTCOME MEASURES Best-corrected visual acuity, kinetic visual field, nystagmus testing, and pupillary light reflex. RESULTS The data showed a statistically significant improvement of best-corrected visual acuity between baseline and 3 years after treatment in the treated eye (P<0.001). In all patients, an enlargement of the area of visual field was observed that remained stable until 3 years after injection (average values: baseline, 1058 deg(2) vs. 3 years after treatment, 4630 deg(2)) and a reduction of the nystagmus frequency compared with baseline at the 3-year time point. Furthermore, a statistically significant difference was observed in the pupillary constriction of the treated eye (P<0.05) compared with the untreated eye in 3 patients at 1- and 3-year time points. No patients experienced serious adverse events related to the vector in the 3-year postinjection period. CONCLUSIONS The long-term follow-up data (3 years) on the 5-patient Italian cohort involved in the LCA2 gene therapy clinical trial clearly showed a stability of improvement in visual and retinal function that had been achieved a few months after treatment. Longitudinal data analysis showed that the maximum improvement was achieved within 6 months after treatment, and the visual improvement was stable up to the last observed time point. FINANCIAL DISCLOSURE(S) Proprietary or commercial disclosure may be found after the references.

Cannabinoid CB2 receptor activation reduces mouse myocardial ischemia‐reperfusion injury: involvement of cytokine/chemokines and PMN

In this study, we have assessed the activation of the cannabinoid CB2 receptor (CB2‐R) in a model of mouse myocardial ischemia/reperfusion (I/R). The results show that treatment of animals with WIN55212‐2, a CB1/CB2‐R agonist, given 30 min before induction of I/R, significantly reduced the extent of infarct size (IS) in the area at risk, as measured 2.5 h later, with almost a 51% inhibition observed at the dose tested of 3.5 mg/kg intraperitoneally (i.p.). The protective effect of WIN55212‐2 was almost abolished by the selective CB2‐R antagonist AM630 (1 mg/kg i.p.) and not affected by the selective CB1‐R antagonist AM251 (3 mg/kg i.p.). The CB2‐R antagonist administered alone produced a slight but significant (P<0.05) increase in IS compared with vehicle alone. The protection afforded by WIN55212‐2 was paralleled by lower values of myeloperoxidase activity and interleukin‐1β and of the CXC chemokine ligand 8 into the injured tissue. In conclusion, we demonstrate for the first time that exogenous and endogenous CB2‐R activation reduces the leukocyte‐dependent myocardial damage associated with an I/R procedure.

Effective delivery of large genes to the retina by dual AAV vectors

Retinal gene therapy with adeno‐associated viral (AAV) vectors is safe and effective in humans. However, AAVs limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardts disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAVs ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans‐splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans‐splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans‐splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.

AAV-mediated photoreceptor transduction of the pig cone-enriched retina

Recent success in clinical trials supports the use of adeno-associated viral (AAV) vectors for gene therapy of retinal diseases caused by defects in the retinal pigment epithelium (RPE). In contrast, evidence of the efficacy of AAV-mediated gene transfer to retinal photoreceptors, the major site of inherited retinal diseases, is less robust. In addition, although AAV-mediated RPE transduction appears efficient, independently of the serotype used and species treated, AAV-mediated photoreceptor gene transfer has not been systematically investigated thus so far in large animal models, which also may allow identifying relevant species-specific differences in AAV-mediated retinal transduction. In the present study, we used the porcine retina, which has a high cone/rod ratio. This feature allows to properly evaluate both cone and rod photoreceptors transduction and compare the transduction characteristics of AAV2/5 and 2/8, the two most efficient AAV vector serotypes for photoreceptor targeting. Here we show that AAV2/5 and 2/8 transduces both RPE and photoreceptors. AAV2/8 infects and transduces photoreceptor more efficiently than AAV2/5, similarly to what we have observed in the murine retina. The use of the photoreceptor-specific rhodopsin promoter restricts transgene expression to porcine rods and cones, and results in photoreceptor transduction levels similar to those obtained with the ubiquitous promoters tested. Finally, immunological, toxicological and biodistribution studies support the safety of AAV subretinal administration to the large porcine retina. The data presented here on AAV-mediated transduction of the cone-enriched porcine retina may affect the development of gene-based therapies for rare and common severe photoreceptor diseases.

Effects of nebivolol on human platelet aggregation

It has been documented that &bgr;-adrenergic antagonists can influence platelet aggregation by a mechanism independent of their ability to antagonize &bgr;-adrenoceptors. Nebivolol, a selective &bgr; 1 -adrenergic receptor antagonist with additional hemodynamic effects, is able to vasodilate human forearm vasculature by acting on the l -arginine/nitric oxide pathway. Constitutive nitric oxide synthase is present also in human platelets, resulting in the formation of nitric oxide, an endogenous inhibitor of platelet aggregation. The aim of this study was to investigate the effects of nebivolol on platelet aggregation and in particular to determine the involvement of the platelet l -arginine/nitric oxide pathway. Propranolol, a nonselective &bgr;-adrenergic antagonist, and carvedilol, a &bgr;-blocker with vasodilating properties, were compared with nebivolol on platelet activity. Plasma from healthy male subjects was used. Platelet aggregation was achieved with adenosine diphosphate (ADP) (3 &mgr;M) and collagen (1 &mgr;g/ml), using the Born turbidimetric method to measure platelet aggregation. Our results showed that nebivolol, propranolol, and carvedilol all had an inhibitory effect on both ADP- and collagen-induced platelet aggregation. Nebivolol exhibited the greatest inhibition effect on platelet aggregation. The mechanism responsible for the inhibitory effect of nebivolol appeared to involve a nitric oxide–dependent pathway. Indeed, l -arginine augmented the inhibitory effects of nebivolol on platelet aggregation induced by collagen and ADP. Furthermore, the inhibitory effect of nebivolol on platelet aggregation was reduced in the presence of the nitric oxide synthase inhibitor N G -monomethyl- l -arginine ( l -NMMA). In conclusion, we have demonstrated in this study that nebivolols mechanism of platelet aggregation inhibition differs from that of other &bgr;-adrenergic antagonists by being partially dependent on nitric oxide production.

MicroRNA-Restricted Transgene Expression in the Retina

Background Gene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions. Methodology/Principal Findings To this end, we generated AAV2/5 vectors expressing EGFP and containing four tandem copies of miR-124 or miR-204 complementary sequences in the 3′UTR of the transgene expression cassette. These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrate that miR-124 and miR-204 target sequences can efficiently restrict AAV2/5-mediated transgene expression to retinal pigment epithelium and photoreceptors, respectively, in mice and pigs. Interestingly, transgene restriction was observed at low vector doses relevant to therapy. Conclusions We conclude that microRNA-mediated regulation of transgene expression can be applied in the retina to either restrict to a specific cell type the robust expression obtained using ubiquitous promoters or to provide an additional layer of gene expression regulation when using cell-specific promoters.

Genotype-Phenotype Correlation in Italian Families with Stargardt Disease

Autosomal recessive Stargardt disease (STGD) has been associated with substantial genetic and phenotypic heterogeneity. By systematic clinical analyses of STGD patients with complete genetic data (i.e. identified mutations on both alleles of the ABCA4 gene), we set out to determine phenotypic subtypes and to correlate these with specific ABCA4 alleles. Twenty-eight patients from 18 families with STGD/fundus flavimaculatus were investigated. All patients were submitted to complete ophthalmologic examination, electrophysiology, fluorescein angiography and ABCA4 gene chip analysis. Two main clinical phenotypes were observed among the examined patients. The severe phenotype was characterized by the onset of the disease <20 years and reduced ERG response, whereas the mild phenotype presented with later onset of the disease and a normal ERG response. Genetic analysis of the ABCA4 gene revealed, in the severe group, more frequently deletions, stop codons and insertions as compared to the mild phenotype group (p = 0.0113 by Fisher’s exact test). Moreover, the compound heterozygous mutations G1961E/5018 + 2T → C found in 7 patients from 3 unrelated STGD families were associated with a mild phenotype in all subjects, except 1. This study documented variability of the clinical expression of STGD in relation to the age of onset of the disease, fundus appearance and the ERG response and allowed to subdivide patients into a severe and a mild phenotype group. These findings suggest that an extensive and comprehensive genetic analysis of STGD patients combined with thorough clinical evaluation, including the careful recording of the age of onset of the disease, would allow a more precise prognostic evaluation.