Maria I. Patrício

Inclusion of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances AAV2-Driven Transduction of Mouse and Human Retina

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) has been included in the transgene cassette of adeno-associated virus (AAV) in several gene therapy clinical trials, including those for inherited retinal diseases. However, the extent to which WPRE increases transgene expression in the retina is still unclear. To address this question, AAV2 vectors containing a reporter gene with and without WPRE were initially compared in vitro and subsequently in vivo by subretinal delivery in mice. In both instances, the presence of WPRE led to significantly higher levels of transgene expression as measured by fundus fluorescence, western blot, and immunohistochemistry. The two vectors were further compared in human retinal explants derived from patients undergoing clinically indicated retinectomy, where again the presence of WPRE resulted in an enhancement of reporter gene expression. Finally, an analogous approach using a transgene currently employed in a clinical trial for choroideremia delivered similar results both in vitro and in vivo, confirming that the WPRE effect is transgene independent. Our data fully support the inclusion of WPRE in ongoing and future AAV retinal gene therapy trials, where it may allow a therapeutic effect to be achieved at an overall lower dose of vector.

Retinal Degeneration in Choroideremia follows an Exponential Decay Function

Fundus autofluorescence (AF) arises from lipofuscin, which is derived from retinoid byproducts of the visual cycle and accumulates within retinal pigment epithelial cells. Alterations in AF pattern are seen in a wide range of retinal degenerations. Choroideremia is an X-linked retinal dystrophy caused by loss-of-function mutations within the CHM gene, encoding Rab escort protein-1. It is uniquely characterized by a central “island” of residual AF that undergoes gradual shrinkage with disease progression. The decrease in AF area is correlated precisely with loss of overlying photoreceptors, leading to progressive visual field restriction and blindness around the fifth decade. Retinal gene replacement therapy using an adeno-associated viral vector could potentially slow down or stop disease progression in choroideremia. Although visual acuity may be improved by gene therapy, it is affected relatively late in the disease; therefore, the area of residual AF may provide an alternative anatomic biomarker for monitoring progression at earlier stages. A previous cross-sectional study suggested an exponential decrease in AF area with age in choroideremia. However, longitudinal data on natural disease progression is lacking. For instance, it is uncertain whether individuals progressed at different rates depending on genetic, epigenetic, or environmental factors, and whether the rate of progression varied between early and late stages of the disease.

The Biological Activity of AAV Vectors for Choroideremia Gene Therapy Can Be Measured by In Vitro Prenylation of RAB6A

Choroideremia (CHM) is a rare, X-linked recessive retinal dystrophy caused by mutations in the CHM gene. CHM is ubiquitously expressed in human cells and encodes Rab escort protein 1 (REP1). REP1 plays a key role in intracellular trafficking through the prenylation of Rab GTPases, a reaction that can be reproduced in vitro. With recent advances in adeno-associated virus (AAV) gene therapy for CHM showing gene replacement to be a promising approach, an assay to assess the biological activity of the vectors is of the uttermost importance. Here we sought to compare the response of two Rab proteins, RAB27A and RAB6A, to the incorporation of a biotinylated lipid donor in a prenylation reaction in vitro. First, we found the expression of REP1 to be proportional to the amount of recombinant AAV (rAAV)2/2-REP1 used to transduce the cells. Second, prenylation of RAB6A appeared to be more sensitive to REP1 protein expression than prenylation of RAB27A. Moreover, the method was reproducible in other cell lines. These results support the further development of a prenylation reaction using a biotinylated lipid donor and RAB6A to assess the biological activity of AAV vectors for CHM gene therapy.

Impact of Vital Dyes on Cell Viability and Transduction Efficiency of AAV Vectors Used in Retinal Gene Therapy Surgery: An In Vitro and In Vivo Analysis

Purpose Treatment of inherited retinal degenerations using adeno-associated viral (AAV) vectors involves delivery by subretinal injection. In the latter stages, alteration of normal anatomy may cause difficulty in visualizing the retinotomy, retinal detachment extension, and vector diffusion. Vital dyes may be useful surgical adjuncts, but their safety and impact on AAV transduction are largely unknown. Methods The effects of Sodium Fluorescein (SF), Membrane Blue (MB), and Membrane Blue Dual (DB) at a range of dilutions were assessed on human embryonic kidney cells in vitro using an AAV2-green fluorescent protein (GFP) reporter at different multiplicities of infection. Flow cytometry analysis was performed to assess both cell viability and transduction efficiency. The effect on quantitative (q)PCR titer was determined. Balanced salt solution (BSS) or dilute DB (1:5 in BSS) were delivered subretinally into left/right eyes of C57BL/6J mice (n = 12). Retinal structure and function were analyzed by optical coherence tomography, autofluorescence, dark-and light-adapted full-field electroretinography. Results DB and MB were not toxic at any concentration tested, SF only when undiluted. The presence of dyes did not adversely affect the genomic titer. DB even increased the values, due to presence of surfactant in the formulation. AAV2-GFP transduction efficiency was not reduced by the dyes. No structural and functional toxic effects were observed following subretinal delivery of DB. Conclusions Only undiluted SF affected cell viability. No effects on qPCR titer and transduction efficiency were observed. DB does not appear toxic when delivered subretinally and improves titer accuracy. DB may therefore be a safe and helpful adjunct during gene therapy surgery. Translational Relevance This paper might be of interest to the retinal gene therapy community: it is a “bench to bedside” research paper about the potential use of dyes as a surgical adjunct during the gene therapy surgery. We have tested the potential toxicity and impact on transduction efficiency in an in vitro and in vivo model.

A clinical-grade gene therapy vector for pharmacoresistant epilepsy successfully overexpresses NPY in a human neuronal cell line

PURPOSE Epilepsy is a common neurological condition characterised by recurrent unprovoked seizures and often treatable with appropriate medication. However, almost 30% of cases are pharmacoresistant and while a proportion of these may be amenable to resective surgery, a gene therapy approach could be an attractive alternative option. Neuropeptide Y (NPY) has anticonvulsant and anti-epileptogenic properties in animal models of temporal lobe epilepsy when delivered by an adeno-associated viral (AAV) vector. Here we sought to demonstrate successful secretion of NPY from AAV-transduced human neuronal cells, which would be essential in planning any clinical trial. METHODS A human neuroblastoma cell line (SH-SY5Y) was used to assess in vitro whether an AAV vector manufactured to clinical-grade protocols would be effective at transducing these cells to express NPY. Optimal transduction efficiency was first achieved with retinoic acid and tetradecanoylphorpol-13-acetate (TPA) treatment, prior to expose to AAV1-green fluorescent protein (GFP) reporter vector, AAV1-NPY therapeutic vector or sham treated with no vector. Levels of NPY in cell supernatants were determined using two antibody-based methods RESULTS: We found that the levels of NPY released into the cell culture media supernatant, and protein extracts of the cell pellet, were significantly higher following exposure to AAV1-NPY than when compared to either a control GFP reporter vector (AAV1-GFP) or sham treated controls. CONCLUSION This first demonstration that an AAV-NPY construct can successfully transduce human neuronal cells supports the pre-clinical development of a clinical trial using AAV-based NPY for pharmacoresistant epilepsy.

Stroma-derived IL-6, G-CSF and Activin-A mediated dedifferentiation of lung carcinoma cells into cancer stem cells.

Cancer stem cells (CSCs) are a small population of resistant cells inhabiting the tumors. Although comprising only nearly 3% of the tumor mass, these cells were demonstrated to orchestrate tumorigenesis and differentiation, underlie tumors’ heterogeneity and mediate therapy resistance and tumor relapse. Here we show that CSCs may be formed by dedifferentiation of terminally differentiated tumor cells under stress conditions. Using a elegant co-culture cellular system, we were able to prove that nutrients and oxygen deprivation activated non-malignant stromal fibroblasts, which in turn established with tumor cells a paracrine loop mediated by Interleukine-6 (IL-6), Activin-A and Granulocyte colony-stimulating factor (G-CSF), that drove subsequent tumor formation and cellular dedifferentiation. However, by scavenging these cytokines from the media and/or blocking exosomes’ mediated communication it was possible to abrogate dedifferentiation thus turning these mechanisms into potential therapeutic targets against cancer progression.

Human Retinal Explant Culture for Ex Vivo Validation of AAV Gene Therapy

Recombinant adeno-associated viral (AAV) vectors have been successfully employed as the mode of gene delivery in several clinical trials for the treatment of inherited retinal diseases to date. The design of such vectors is critical in determining cellular tropism and level of subsequent gene expression that may be achieved following viral delivery. Here we describe a system for living retinal tissue extraction, ex vivo culture, viral transduction and assessment of transgene expression that may be used to assess viral constructs for gene therapy in the human retina at a preclinical stage.

Retinal Gene Therapy for Choroideremia: In Vitro Testing for Gene Augmentation Using an Adeno-Associated Viral (AAV) Vector

As gene therapy of choroideremia is becoming a clinical reality, there is a need for reliable and sensitive assays to determine the expression of exogenously delivered Rab Escort Protein-1 (REP1), in particular to test new gene therapy vectors and as a quality control screen for clinical vector stocks. Here we describe an in vitro protocol to test transgene expression following AAV2/2-REP1 transduction of a human cell line. Gene augmentation can be confirmed by western blot and quantification of the fold-increase of human REP1 levels over untransduced controls.

Beneficial effects on vision in patients undergoing retinal gene therapy for choroideremia

Retinal gene therapy is increasingly recognized as a novel molecular intervention that has huge potential in treating common causes of blindness, the majority of which have a genetic aetiology1–5. Choroideremia is a chronic X-linked retinal degeneration that was first described in 18726. It leads to progressive blindness due to deficiency of Rab-escort protein 1 (REP1). We designed an adeno-associated viral vector to express REP1 and assessed it in a gene therapy clinical trial by subretinal injection in 14 patients with choroideremia. The primary endpoint was vision change in treated eyes 2 years after surgery compared to unoperated fellow eyes. Despite complications in two patients, visual acuity improved in the 14 treated eyes over controls (median 4.5 letter gain, versus 1.5 letter loss, P = 0.04), with 6 treated eyes gaining more than one line of vision (>5 letters). The results suggest that retinal gene therapy can sustain and improve visual acuity in a cohort of predominantly late-stage choroideremia patients in whom rapid visual acuity loss would ordinarily be predicted.The long-term follow-up results of a phase 1/2 retinal gene therapy clinical trial for choroideremia (NCT01461213) support the safety and efficacy of the treatment.

Choroideremia: molecular mechanisms and development of AAV gene therapy.

ABSTRACT Introduction: Choroideremia is an X-linked inherited retinal degeneration that causes blindness in afflicted males by middle age. The causative gene, CHM, plays a key role in intracellular trafficking pathways, and its disruption impairs cell homeostasis. Areas covered: The mechanism by which mutations in CHM cause choroideremia is still under debate. Here we describe the molecular defects in choroideremia cells regarding both the deficiency of prenylation and the involvement of Rab GTPases. Important in vivo and in vitro studies that contributed to the current knowledge are also discussed. Finally, the rationale for the development of a treatment strategy using AAV for gene replacement is presented, together with other treatment strategies under consideration. Expert opinion: Despite ubiquitous expression of the CHM gene, the primary defect in choroideremia is driven by retinal pigment epithelium (RPE) and photoreceptors degeneration. Here we discuss how impairment of vesicular trafficking pathways in the RPE plays a major role in the molecular pathogenesis of choroideremia. Moreover, this defect is likely restored by subretinal delivery of a functional copy of CHM using AAV, as evidenced by clinical trial results. The surgical complexity of delivering the AAV vector to the target area remains as the main challenge to this therapy. Abbreviations: AAV: adeno-associated virus; BCD: Bietti’s crystalline dystrophy; CHM: choroideremia; CHML: choroideremia-like; Dfp: days post-fertilization; EMA: European Medicines Agency; ERG: electroretinogram; ETDRS: Early Treatment Diabetic Retinopathy Study; FDA: Food and Drug Administration; FTase: farnesyl transferase; GFP: green fluorescent protein; GGPP: geranylgeranyl-diphosphate; GGTase-I: geranylgeranyl transferase type-I; GGTase-II: geranylgeranyl transferase type-II; HMG-CoA: 3-hydroxy-3-methylglutayl-CoA; HMGCR: HMG-CoA reductase; iPSC: induced pluripotent stem cells; IRDs: inherited retinal diseases; KO: knockout; LCA: Leber congenital amaurosis; NMD: nonsense-mediated mRNA decay; OCT: optical coherence tomography; PMBCs: peripheral blood mononuclear cells; POS: photoreceptor outer segments; PTCs: premature termination codons; Rab GGTase: Rab geranylgeranyl transferase; REP: Rab escort protein; RPE: retinal pigment epithelium; TRIDs: translational read-through inducing drugs; WPRE: woodchuck post-transcriptional regulatory element