Eric A. Pierce

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.

The Proteome of the Mouse Photoreceptor Sensory Cilium Complex

Primary cilia play critical roles in many aspects of biology. Specialized versions of primary cilia are involved in many aspects of sensation. The single photoreceptor sensory cilium (PSC) or outer segment elaborated by each rod and cone photoreceptor cell of the retina is a classic example. Mutations in genes that encode cilia components are common causes of disease, including retinal degenerations. The protein components of mammalian primary and sensory cilia have not been defined previously. Here we report a detailed proteomics analysis of the mouse PSC complex. The PSC complex comprises the outer segment and its cytoskeleton, including the axoneme, basal body, and ciliary rootlet, which extends into the inner segment of photoreceptor cells. The PSC complex proteome contains 1968 proteins represented by three or more unique peptides, including ∼1500 proteins not detected in cilia from lower organisms. This includes 105 hypothetical proteins and 60 proteins encoded by genes that map within the critical intervals for 23 inherited cilia-related disorders, increasing their priority as candidate genes. The PSC complex proteome also contains many cilia proteins not identified previously in photoreceptors, including 13 proteins produced by genes that harbor mutations that cause cilia disease and seven intraflagellar transport proteins. Analyses of PSC complexes from rootletin knock-out mice, which lack ciliary rootlets, confirmed that 1185 of the identified PSC complex proteins are derived from the outer segment. The mass spectrometry data, benchmarked by 15 well characterized outer segment proteins, were used to quantify the copy number of each protein in a mouse rod outer segment. These results reveal mammalian cilia to be several times more complex than the cilia of unicellular organisms and open novel avenues for studies of how cilia are built and maintained and how these processes are disrupted in human disease.

Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

ABSTRACT Specific mRNA degradation mediated by double-stranded RNA (dsRNA) interference (RNAi) is a powerful way of suppressing gene expression in plants, nematodes, and fungal, insect, and protozoan systems. However, only a few cases of RNAi have been reported in mammalian systems. Here, we investigated the feasibility of the RNAi strategy in several mammalian cells by using the enhanced green fluorescent protein gene as a target, either by in situ production of dsRNA from transient transfection of a plasmid harboring a 547-bp inverted repeat or by direct transfection of dsRNA made by in vitro transcription. Several mammalian cells including differentiated embryonic stem (ES) cells did not exhibit specific RNAi in transient transfection. This long dsRNA, however, was capable of inducing a sequence-specific RNAi for the episomal and chromosomal target gene in undifferentiated ES cells. dsRNA at 8.3 nM decreased the cognate gene expression up to 70%. However, RNAi activity was not permanent because it was more pronounced in early time points and diminished 5 days after transfection. Thus, undifferentiated ES cells may lack the interferon response, similar to mouse embryos and oocytes. Regardless of their apparent RNAi activity, however, cytoplasmic extracts from mammalian cells produced a small RNA of 21 to 22 nucleotides from the long dsRNA. Our results suggest that mammalian cells may possess RNAi activity but nonspecific activation of the interferon response by longer dsRNA may mask the specific RNAi. The findings offer an opportunity to use dsRNA for inhibition of gene expression in ES cells to study differentiation.

AAV2 Gene Therapy Readministration in Three Adults with Congenital Blindness

Repeat administration of gene therapy to the contralateral retina of three congenitally blind patients was safe and resulted in improved vision. Shining a Light with Gene Therapy Gene therapy has great potential for treating certain diseases by providing therapeutic genes to target cells. Administration of a gene therapy vector carrying the RPE65 gene in 12 patients with congenital blindness due to RPE65 mutations led to improvements in retinal and visual function and proved to be a safe and stable procedure. In a follow-up study, the same group of researchers led by Jean Bennett set out to discover whether it would be possible to safely administer the vector and the therapeutic transgene to the contralateral eye of the patients. A big concern was whether the first gene therapy injection might have primed the patients’ immune system to respond to the adeno-associated virus (AAV) vector or the product of the therapeutic transgene that it had delivered. To test the safety and efficacy of a second administration of gene therapy to the second eye, the authors demonstrated that readministration was both safe and effective in animal models. Then, they selected 3 of the original 12 patients and readministered the AAV vector and its RPE65 transgene to the contralateral eye. They assessed safety by evaluating inflammatory responses, immune reactions, and extraocular exposure to the AAV vector. Efficacy was assessed through qualitative and quantitative measures of retinal and visual function including the ability to read letters, the extent of side vision, light sensitivity, the pupillary light reflex, the ability to navigate in dim light, and evidence from neuroimaging studies of cortical activation (which demonstrated that signals from the retina were recognized by the brain). The researchers did not discover any safety concerns and did not identify harmful immune responses to the vector or the transgene product. Before and after comparisons of psychophysical data and cortical responses provided the authors with evidence that gene therapy readministration was effective and mediated improvements in retinal and visual function in the three patients. The researchers report that the lack of immune response and the robust safety profile in this readministration gene therapy study may be due in part to the immune-privileged nature of the eye, and the low dose and very pure preparation of the AAV vector. Demonstration of safe and stable reversal of blindness after a single unilateral subretinal injection of a recombinant adeno-associated virus (AAV) carrying the RPE65 gene (AAV2-hRPE65v2) prompted us to determine whether it was possible to obtain additional benefit through a second administration of the AAV vector to the contralateral eye. Readministration of vector to the second eye was carried out in three adults with Leber congenital amaurosis due to mutations in the RPE65 gene 1.7 to 3.3 years after they had received their initial subretinal injection of AAV2-hRPE65v2. Results (through 6 months) including evaluations of immune response, retinal and visual function testing, and functional magnetic resonance imaging indicate that readministration is both safe and efficacious after previous exposure to AAV2-hRPE65v2.

TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum

Ciliary dysfunction leads to a broad range of overlapping phenotypes, collectively termed ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifier alleles to clinically distinct disorders. Here we show that mutations in TTC21B, which encodes the retrograde intraflagellar transport protein IFT139, cause both isolated nephronophthisis and syndromic Jeune asphyxiating thoracic dystrophy. Moreover, although resequencing of TTC21B in a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations showed a significant enrichment of pathogenic alleles in cases (P < 0.003), suggesting that TTC21B contributes pathogenic alleles to ∼5% of ciliopathy cases. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies and interact in trans with other disease-causing genes and highlight how saturated resequencing followed by functional analysis of all variants informs the genetic architecture of inherited disorders.

Comparative analysis of RNA-Seq alignment algorithms and the RNA-Seq unified mapper (RUM)

MOTIVATION A critical task in high-throughput sequencing is aligning millions of short reads to a reference genome. Alignment is especially complicated for RNA sequencing (RNA-Seq) because of RNA splicing. A number of RNA-Seq algorithms are available, and claim to align reads with high accuracy and efficiency while detecting splice junctions. RNA-Seq data are discrete in nature; therefore, with reasonable gene models and comparative metrics RNA-Seq data can be simulated to sufficient accuracy to enable meaningful benchmarking of alignment algorithms. The exercise to rigorously compare all viable published RNA-Seq algorithms has not been performed previously. RESULTS We developed an RNA-Seq simulator that models the main impediments to RNA alignment, including alternative splicing, insertions, deletions, substitutions, sequencing errors and intron signal. We used this simulator to measure the accuracy and robustness of available algorithms at the base and junction levels. Additionally, we used reverse transcription-polymerase chain reaction (RT-PCR) and Sanger sequencing to validate the ability of the algorithms to detect novel transcript features such as novel exons and alternative splicing in RNA-Seq data from mouse retina. A pipeline based on BLAT was developed to explore the performance of established tools for this problem, and to compare it to the recently developed methods. This pipeline, the RNA-Seq Unified Mapper (RUM), performs comparably to the best current aligners and provides an advantageous combination of accuracy, speed and usability. AVAILABILITY The RUM pipeline is distributed via the Amazon Cloud and for computing clusters using the Sun Grid Engine (http://cbil.upenn.edu/RUM). CONTACT ggrant@pcbi.upenn.edu; epierce@mail.med.upenn.edu SUPPLEMENTARY INFORMATION The RNA-Seq sequence reads described in the article are deposited at GEO, accession GSE26248.

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.

Mutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pigmentosa.

The autosomal dominant retinitis pigmentosa (RP) locus, designated RP1, has been mapped through linkage studies to a 4-cM interval at 8q11–13. Here we describe a new photoreceptor-specific gene that maps in this interval and whose expression is modulated by retinal oxygen levels in vivo. This gene consists of at least 4 exons that encode a predicted protein of 2,156 amino acids. A nonsense mutation at codon 677 of this gene is present in approximately 3% of cases of dominant RP in North America. We also detected two deletion mutations that cause frameshifts and introduce premature termination codons in three other families with dominant RP. Our data suggest that mutations in this gene cause dominant RP, and that the encoded protein has an important but unknown role in photoreceptor biology.