Lentiviral vector (LVV)-mediated cell and gene therapies have the potential to cure diseases that currently require lifelong intervention. However, the requirement for plasmid transfection hinders large-scale LVV manufacture. Moreover, large-scale plasmid production, testing, and transfection contribute to operational risk and the high cost associated with this therapeutic modality.
View Article and Find Full Text PDFRecombinant 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.
View Article and Find Full Text PDFPurpose: 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.
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.
View Article and Find Full Text PDFMutations 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.
View Article and Find Full Text PDFExpert Rev Ophthalmol
December 2019
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 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.
View Article and Find Full Text PDFImportance: 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.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFMutations in the human 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 Ca-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.
View Article and Find Full Text PDFChoroideremia 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.
View Article and Find Full Text PDFRetinal 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 aetiology. Choroideremia is a chronic X-linked retinal degeneration that was first described in 1872. It leads to progressive blindness due to deficiency of Rab-escort protein 1 (REP1).
View Article and Find Full Text PDFCancer 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.
View Article and Find Full Text PDFExpert Opin Biol Ther
July 2018
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.
Choroideremia (CHM) is a rare, X-linked recessive retinal dystrophy caused by mutations in the gene. 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 .
View Article and Find Full Text PDFPurpose: 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.
View Article and Find Full Text PDFRecombinant 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.
View Article and Find Full Text PDFAs 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.
View Article and Find Full Text PDFPurpose: 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.
View Article and Find Full Text PDFThe 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.
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