Erratum: Enhancement of Adeno-Associated Virus-Mediated Gene Therapy Using Hydroxychloroquine in Murine and Human Tissues.

[This corrects the article DOI: 10.1016/j.omtm.2019.05.012.].

Self-attenuating adenovirus enables production of recombinant adeno-associated virus for high manufacturing yield without contamination.

Recombinant adeno-associated virus (rAAV) shows great promise for gene therapy, however scalability, yield and quality remain significant issues. Here we describe an rAAV manufacturing strategy using a 'helper' adenovirus that self-inhibits its major late promoter (MLP) to truncate its own replication. Inserting a tetracycline repressor (TetR) binding site into the MLP and encoding the TetR under its transcriptional control allowed normal adenovirus replication in the presence of doxycycline but only genome amplification and early gene expression (the 'helper' functions) in its absence. Using this self-inhibiting adenovirus we demonstrate delivery of adenoviral helper functions, AAV rep and cap genes, and the rAAV genome to yield up to 30-fold more rAAV vectors compared to the helper-free plasmid approach and significant improvements in particle infectivity for a range of serotypes. This system allows significant improvements in the production of serotypes rAAV2, rAAV6, rAAV8 and rAAV9, and enables propagation of existing rAAV without transfection, a process that improves batch quality by depleting reverse packaged DNA contaminants. We propose this as a high-yielding, contaminant-free system suitable for scalable rAAV manufacture.

Expression of Rab Prenylation Pathway Genes and Relation to Disease Progression in Choroideremia.

Purpose: Choroideremia results from the deficiency of Rab Escort Protein 1 (REP1), encoded by CHM, involved in the prenylation of Rab GTPases. Here, we investigate whether the transcription and expression of other genes involved in the prenylation of Rab proteins correlates with disease progression in a cohort of patients with choroideremia. Methods: Rates of retinal pigment epithelial area loss in 41 patients with choroideremia were measured using fundus autofluorescence imaging for up to 4 years. From lysates of cultured skin fibroblasts donated by patients (n = 15) and controls (n = 14), CHM, CHML, RABGGTB and RAB27A mRNA expression, and REP1 and REP2 protein expression were compared. Results: The central autofluorescent island area loss in patients with choroideremia occurred with a mean half-life of 5.89 years (95% confidence interval [CI] = 5.09-6.70), with some patients demonstrating relatively fast or slow rates of progression (range = 3.3-14.1 years). Expression of CHM mRNA and REP1 protein were significantly decreased in all patients. No difference in expression of CHML, RABGGTB, RAB27A, or REP2 was seen between patients and controls. No correlation was seen between expression of the genes analyzed and rates of retinal degeneration. Non-sense induced transcriptional compensation of CHML, a CHM-like retrogene, was not observed in patients with CHM variants predicted to undergo non-sense mediated decay. Conclusions: Patients with choroideremia, who are deficient for REP1, show normal levels of expression of other genes involved in Rab prenylation, which do not appear to play any modifying role in the rate of disease progression. Translational Relevance: There remains little evidence for selection of patients for choroideremia gene therapy based on genotype.

Analysis of Early Cone Dysfunction in an In Vivo Model of Rod-Cone Dystrophy.

Retinitis pigmentosa (RP) is a generic term for a group of genetic diseases characterized by loss of rod and cone photoreceptor cells. Although the genetic causes of RP frequently only affect the rod photoreceptor cells, cone photoreceptors become stressed in the absence of rods and undergo a secondary degeneration. Changes in the gene expression profile of cone photoreceptor cells are likely to occur prior to observable physiological changes. To this end, we sought to achieve greater understanding of the changes in cone photoreceptor cells early in the degeneration process of the Rho-/- mouse model. To account for gene expression changes attributed to loss of cone photoreceptor cells, we normalized PCR in the remaining number of cones to a cone cell reporter (OPN1-GFP). Gene expression profiles of key components involved in the cone phototransduction cascade were correlated with tests of retinal cone function prior to cell loss. A significant downregulation of the photoreceptor transcription factor Crx was observed, which preceded a significant downregulation in cone opsin transcripts that coincided with declining cone function. Our data add to the growing understanding of molecular changes that occur prior to cone dysfunction in a model of rod-cone dystrophy. It is of interest that gene supplementation of CRX by adeno-associated viral vector delivery prior to cone cell loss did not prevent cone photoreceptor degeneration in this mouse model.

Effect of AAV-Mediated Rhodopsin Gene Augmentation on Retinal Degeneration Caused by the Dominant P23H Rhodopsin Mutation in a Knock-In Murine Model.

Mutations in the rhodopsin gene may cause photoreceptor degeneration in autosomal dominant retinitis pigmentosa (ADRP) by dominant negative or toxic gain-of-function mechanisms. Controversy exists as to the mechanism by which the widely studied P23H mutation induces rod cell dysfunction and death. Inherited disease caused by dominant negative mutations may be amenable to treatment using wild-type gene augmentation. Indeed, prior studies in the RHOP23H, Rho+/- transgenic mouse model of ADRP have suggested that a therapeutic benefit may be achieved when wild-type rhodopsin is overexpressed following subretinal delivery of a recombinant adeno-associated viral (AAV) vector. In this study, we investigated the effect of wild-type rhodopsin supplementation on the rate of retinal degeneration in the more clinically relevant RhoP23H/+ knock-in mouse model of ADRP. Four AAVs carrying the human rhodopsin coding sequence were first designed and compared for efficacy in the rhodopsin knockout mouse. All four vectors were capable of driving expression of the human transgene in the knockout retina with the protein being appropriately trafficked to de novo rod outer segments. The most efficient of these vectors was injected at one of two doses into the subretinal space of RhoP23H/+ mice and the effect on retinal structure and function determined longitudinally by spectral-domain optical coherence tomography and electroretinography, respectively, over a 3-month period. Although significant overexpression of rhodopsin protein was achieved in this model, no beneficial effect on retinal structure or function was observed at either dose. Lack of therapeutic efficacy in this model may be attributable to the relative rapidity of degeneration in the RhoP23H/+ mouse relative to the human disease, over- or under dosing at the level of individual photoreceptors, late timing of the intervention, or a possible predominant toxic gain-of-function mechanism of degeneration.

Inclusion of PF68 Surfactant Improves Stability of rAAV Titer when Passed through a Surgical Device Used in Retinal Gene Therapy.

Recent advances in recombinant adeno-associated virus (rAAV) gene therapy for choroideremia show gene replacement to be a promising approach. It is, however, well known that contact of vector solution with plastic materials in the surgical device may result in non-specific adsorption with resulting loss of physical titer and/or level of protein expression and activity. Here we assessed the biocompatibility and stability of rAAV2-REP1 (Rab Escort Protein-1) before and following passage through the injection device over a period of time to mimic the clinical scenario. Three identical devices were screened using two concentrations of vector: high (1E+12 DNase-resistant particles [DRP]/mL) and low (1E+11 DRP/mL), to mimic high- and low-dose administrations of vector product. The low dose was prepared using either formulation buffer that contained 0.001% of a non-ionic surfactant (PF68) or balanced salt solution (BSS). We observed significant losses in the genomic titer of samples diluted with BSS for all time points. The addition of 0.001% PF68 did not, however, affect rAAV physical titer, or REP1 protein expression and biological activity. Hence we observed that neither the genomic titer nor the biological activity of a rAAV2-REP1-containing solution was affected following passage through the surgical device when PF68 was present as a surfactant and this was maintained over a period up to 10 h.

Association of Messenger RNA Level With Phenotype in Patients With Choroideremia: Potential Implications for Gene Therapy Dose.

Importance: Gene therapy is a promising treatment for choroideremia, an X-linked retinal degeneration. The required minimum level of gene expression to ameliorate degeneration rate is unknown. This can be interrogated by exploring the association between messenger RNA (mRNA) levels and phenotype in mildly affected patients with choroideremia. Objective: To analyze CHM mRNA splicing outcomes in 2 unrelated patients with the same c.940+3delA CHM splice site variant identified as mildly affected from a previous study of patients with choroideremia. Design, Setting, and Participants: In this retrospective observational case series, 2 patients with c.940+3delA CHM variants treated at a single tertiary referral center were studied. In addition, a third patient with a c.940+2T>A variant that disrupts the canonical dinucleotide sequence at the same donor site served as a positive control. Data were collected from October 2013 to July 2018. Main Outcomes and Measures: Central area of residual fundus autofluorescence was used as a biomarker for disease progression. CHM transcript splicing was assessed by both end point and quantitative polymerase chain reaction. Rab escort protein 1 (REP1) expression was assessed by immunoblot. Results: The 2 mildly affected patients with c.940+3delA variants had large areas of residual autofluorescence for their age and longer degeneration half-lives compared with the previous cohort of patients with choroideremia. The control patient with a c.940+2T>A variant had a residual autofluorescence area within the range expected for his age. Both patients with the c.940+3delA variant expressed residual levels of full-length CHM mRNA transcripts relative to the predominant truncated transcript (mean [SEM] residual level: patient 1, 2.3% [0.3]; patient 2, 4.7% [0.2]), equivalent to approximately less than 1% of the level of full-length CHM expressed in nonaffected individuals. Full-length CHM expression was undetectable in the control patient. REP1 expression was less than the threshold for detection both in patients 1 and 2 and the control patient compared with wild-type controls. Conclusions and Relevance: These results demonstrate the first genotype-phenotype association in choroideremia. A +3 deletion in intron 7 is sufficient to cause choroideremia in a milder form. If replicated with gene therapy, these findings would suggest that relatively low expression (less than 1%) of the wild-type levels of mRNA would be sufficient to slow disease progression.

Enhancement of Adeno-Associated Virus-Mediated Gene Therapy Using Hydroxychloroquine in Murine and Human Tissues.

The therapeutic effects of gene therapy using adeno-associated virus (AAV) vectors are dependent on the efficacy of viral transduction. Currently, we have reached the safe limits of AAV vector dose, beyond which damaging inflammatory responses are seen. To improve the efficacy of AAV transduction, we treated mouse embryonic fibroblasts, primate retinal pigment epithelial cells, and human retinal explants with hydroxychloroquine (HCQ) 1 h prior to transduction with an AAV2 vector encoding GFP driven by a ubiquitous CAG promoter. This led to a consistent increase in GFP expression, up to 3-fold, compared with vector alone. Comparing subretinal injections of AAV2.CAG.GFP vector alone versus co-injection with 18.75 µM HCQ in paired eyes in mice, mean GFP expression was 4.6-fold higher in retinae co-treated with HCQ without retinal toxicity. A comparative 5.9-fold effect was seen with an AAV8(Y733F).GRK1.GFP vector containing the photoreceptor-specific rhodopsin kinase promoter. While the mechanism of action remains to be fully elucidated, our data suggest that a single pulse of adjunctive HCQ could safely improve AAV transduction in vivo, thus providing a novel strategy for enhancing the clinical effects of gene therapy.

A Quantitative Chloride Channel Conductance Assay for Efficacy Testing of AAV.BEST1.

Mutations in the human BEST1 gene are responsible for a number of distinct retinal disorders known as bestrophinopathies, for which there are no current treatments. The protein product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE) where it localizes to the basolateral membrane and acts as a Ca2+-activated chloride channel. Recent studies have shown successful BEST1-mediated gene transfer to the RPE, indicating human clinical trials of BEST1 gene therapy may be on the horizon. A critical aspect of such trials is the ability to assess the efficacy of vector prior to patient administration. Here, an assay is presented that enables the quantitative assessment of AAV-mediated BEST1 chloride conductance as a measure of vector efficacy. Expression of BEST1 following transduction of HEK293 cells with AAV.BEST1 vectors was confirmed by liquid chromatography, Western blot, and immunocytochemistry. Whole-cell patch-clamp showed increased chloride conductance in BEST1-transduced cells compared to sham-transduced and untransduced controls. Exogenous chloride current correlated to BEST1 expression level, with an enhanced AAV.BEST1.WPRE vector providing higher expression levels of BEST1 and increases in chloride conductance. This study presents in vitro electrophysical quantification of bestrophin-1 following AAV-mediated gene transfer, providing vital functional data on an AAV gene therapy product that will support a future application for regulatory approval.

Atypical choroideremia presenting with early-onset macular atrophy.

Choroideremia is an X-linked recessive retinal degeneration predominantly affecting hemizygous males. It is caused by mutations in the CHM gene that encodes the Rab escort protein-1. Characteristic features include early nyctalopia followed by progressive constriction of peripheral visual fields and sparing of the central vision until late in life with a distinct fundoscopic appearance. We present the case of a 17-year-old male with a c.282delT in exon 4 of CHM that has not previously been reported. Phenotypically this patient presented with an atypical choroideremia phenotype of early central macular degeneration in addition to the classic peripheral fundus characteristic findings.

Progress in the development of novel therapies for choroideremia.

INTRODUCTION: There are no currently approved treatments for choroideremia, an X-linked progressive inherited retinal degeneration that leads to blindness by middle age. Several treatment options are being explored, but with major advances in adeno-associated vector (AAV) gene replacement therapy that has reached phase III clinical trials. AREAS COVERED: In this review we discuss new insights into the clinical phenotyping and genetic testing of choroideremia patients, that aid disease characterisation, progression and patient inclusion into clinical trials. Recent advances in in-vitro studies have resulted in the development of functional assays that can be used to confirm the diagnosis in challenging cases and to quantify vector potency for use in clinical trials. We review the progress in current gene therapy trials and some considerations towards gene therapy approval for the treatment of choroideremia. Lastly, we discuss developments in alternative therapies including optogenetics. EXPERT COMMENTARY: AAV gene replacement therapy is the most promising treatment strategy for choroideremia, that has developed exponentially over the last few years with a phase III clinical trial now underway. Optogenetics is a promising alternative strategy that might be applicable in late stages of degeneration.

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.

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.

Choroideremia: molecular mechanisms and development of AAV gene therapy.

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.

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.

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.

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.

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.

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.