Implementation Of Gene Therapy Research Articles

New Hope: Using Gene Therapy to Treat Rare Neurological Diseases

Gene therapy is a promising treatment approach for rare/orphan neurological diseases that have limited treatment options and no cure. This article provides a brief overview of different types of rare hereditary neurological diseases and discusses existing gene therapy drugs approved for their treatment. Despite challenges associated with the development and implementation of gene therapy, including cost, delivery, and long-term safety and efficacy, the potential benefits of gene therapy make it a compelling area of research for the treatment of rare hereditary neurological diseases.

Heterochromatin extension: a possible cytogenetic fate of primary amenorrhea along with normal karyotype

Primary amenorrhoea (PA) is considered to be one of the challenging and taxing problems for the gynaecologist. Previous studies suggested that different numerical and structural chromosome abnormalities are associated with this. Heterochromatin polymorphisms are considered to be normal variant but considering the recent research on crucial cellular effects of heterochromatin, we have aimed to find out the prevalence of heteromorphism along with other standard chromosomal abnormalities. This was an observational study which was conducted in Diamond Harbour Govt. Medical College and Hospital, West Bengal during March 2019–February 2021. Clinical features of 178 patients were noted and peripheral venous blood was taken following informed consent. This comprehensive study reveals that there are 10.11% of the females among 178 females having a heterochromatin extension which is significantly high. We hence suggest that heteromorphism may be associated with ovarian dysfunction leading to amenorrhoea as the region of heterochromatin acts as a key part in chromosome structure, histone modification and gene regulation. Analysis at the molecular level may be needed to unveil any relationship between heteromorphism and PA. Impact Statement What is already known on this subject? Primary amenorrhoea (PA) is a menstrual abnormality found in females with the prevalence of 1–3%. It may be associated with different types of numerical and structural chromosomal anomalies. Among them Turner’s syndrome (pure and in variant form) is the commonest chromosomal aberration associated with PA. Some patients with PA are found to have a normal karyotype with heterochromatin extension on the large arm (q) of either chromosome 9 or chromosome 16. Chromosomal polymorphism with increase in heterochromatin region consists of highly repetitive sequences of satellite DNA, which normally does not encode any protein and thus considered to be a normal variant. What do the results of this study add? This comprehensive study reveals that there are 10.11% of the females among 178 females having a heterochromatin extension which is significantly high. PA and certain association of phenotypical stigmata like short stature in these patients with heterochromatin extension can be explained on the basis of histone modification and gene regulation by heterochromatin. What are the implications of these findings for clinical practice and/or further research? We will be able to know about involved transcription factors those are responsible for the histone modification directly linked to the heterochromatin extension by further molecular study. That will definitely help to find out the reason for PA as well as implementation of gene therapy in these cases.

Clinical Features and Natural History in a Cohort of Chinese Patients with RPE65-Associated Inherited Retinal Dystrophy

RPE65-associated inherited retinal dystrophy (RPE65-IRD) is an early-onset retinal degeneration. The aim of this study was to describe the clinical features and natural course of this disease in a Chinese patient cohort with RPE65 biallelic variants. Thirty patients from 29 unrelated families with biallelic disease-causing RPE65 variants underwent full ophthalmic examinations. Thirteen were followed up over time. An additional 57 Chinese cases from 49 families were retrieved from the literature to analyze the relationship between best-corrected visual acuity (BCVA) and age. Our 30 patients presented age-dependent phenotypic characteristics. Multiple white dots were a clinical feature of young patients, while maculopathy, epiretinal membrane, and bone spicules were common in adult patients. Among the 84 patients, BCVA declined with age in a nonlinear, positive-acceleration relationship (p < 0.001). All patients older than 40 years met the WHO standard for low vision. Longitudinal observation revealed a slower visual acuity loss in patients younger than 20 years than those in their third or fourth decade of life. Our study detailed the clinical features and natural course of disease in Chinese patients with RPE65-IRD. Our results indicated that these patients have a relatively stable BCVA in childhood and adolescence, but eyesight deteriorates rapidly in the third decade of life. These findings may facilitate the implementation of gene therapy in China.

Frequency and phenotypic characteristics of RPE65 mutations in the Chinese population

BackgroundThe retinoid isomerohydrolase RPE65 has received considerable attention worldwide since a successful clinical gene therapy was approved in 2017 as the first treatment for vision loss associated with RPE65-mediated inherited retinal disease. Identifying patients with RPE65 mutations is a prerequisite to assessing the patients’ eligibility to receive RPE65-targeted gene therapies, and it is necessary to identify individuals who are most likely to benefit from gene therapies. This study aimed to investigate the RPE65 mutations frequency in the Chinese population and to determine the genetic and clinical characteristics of these patients.ResultsOnly 20 patients with RPE65 mutations were identified, and RPE65 mutations were determined to be the 14th most common among all patients with genetic diagnoses. Ten novel variants and two hotspots associated with FAP were identified. A literature review revealed that a total of 57 patients of Chinese origin were identified with pathogenic mutations in the RPE65 gene. The mean best Snellen corrected visual acuity was worse (mean 1.3 ± 1.3 LogMAR) in patients older than 20 years old than in those younger than 15 years old (0.68 ± 0.92 LogMAR). Bone spicule-like pigment deposits (BSLPs) were observed in six patients; they were older than those without BSLP and those with white-yellow dots. Genotype–phenotype analysis revealed that truncating variants seem to lead to a more severe clinical presentation, while best corrected visual acuity testing and fundus changes did not correlate with specific RPE65 variants or mutation types.ConclusionsThis study provides a detailed clinical-genetic assessment of patients with RPE65 mutations of Chinese origin. These results may help to elucidate RPE65 mutations in the Chinese population and may facilitate genetic counseling and the implementation of gene therapy in China.

Gene therapy for inherited metabolic diseases.

Over the last two decades, gene therapy has been successfully translated to many rare diseases. The number of clinical trials is rapidly expanding and some gene therapy products have now received market authorisation in the western world. Inherited metabolic diseases (IMD) are orphan diseases frequently associated with a severe debilitating phenotype with limited therapeutic perspective. Gene therapy is progressively becoming a disease-changing therapeutic option for these patients. In this review, we aim to summarise the development of this emerging field detailing the main gene therapy strategies, routes of administration, viral and non-viral vectors and gene editing tools. We discuss the respective advantages and pitfalls of these gene therapy strategies and review their application in IMD, providing examples of clinical trials with lentiviral or adeno-associated viral gene therapy vectors in rare diseases. The rapid development of the field and implementation of gene therapy as a realistic therapeutic option for various IMD in a short term also require a good knowledge and understanding of these technologies from physicians to counsel the patients at best.

Early signs of longitudinal progressive cone photoreceptor degeneration in achromatopsia

AimsTo characterise longitudinal progressive retinal changes in achromatopsia.MethodsUltrahigh-resolution spectral optical coherence tomography (Copernicus, 3 μm axial resolution) was used to obtain tomograms of the fovea from five children and three...

Studies of patients' thymi aid in the discovery and characterization of immunodeficiency in humans

Studying the molecular and genetic bases of primary immunodeficiency is valuable at several levels. First, such information directly benefits patients in both short- and long-term management. Sophisticated diagnostic tools based on these studies can be used early and lead to appropriate treatment before potentially fatal infections and complications arise. Genotyping is also critical for future development and implementation of gene therapy. Secondly, investigating primary immunodeficiency helps understand the normal immune system in humans. As described in this report, the roles of zeta-associated protein of 70 kDa (Zap-70), CD25, and CD3delta are substantially different in humans when compared with the roles of homologous molecules in other species. Last, information obtained from these studies can be applied to other fields of investigation. Prominent examples for such applications include the intensive effort to design and produce specific inhibitors of Zap-70 and Janus kinase 3 as specific immunosuppressive agents. Most types of primary immunodeficiency in general and severe combined immunodeficiency in particular are rare and therefore cannot be easily studied by using traditional genetic methodology. Instead, biochemical methods were used to explore for candidate genes as was the case in the discovery of Zap-70 deficiency. Critical to the success of these discoveries was the careful analysis of patients' thymus glands. Detection of abnormalities in the thymus in these patients, which preceded identification of the genetic defect, aided in the assessment of the severity and nature of the immune disorder (primary versus secondary). Such assessment is critical before high-risk bone marrow transplantation. Equally important was the contribution of studies of the thymus to the description of novel phenotype of immunodeficiency as clearly demonstrated in defining CD8 lymphocytopenia, Zap-70 deficiency, and CD25 deficiency. Indeed, analysis of the thymus directly pointed to CD25 as candidate gene. Recently, the study of thymocyte-derived transcripts using DNA microarrays was key to discovering CD3delta deficiency. Finally, immunohistochemical analysis of the thymus was critical in pinpointing the roles of Zap-70, CD25, and CD3delta in the development of human T cells.

869. Unique Potential of Gene Therapy for GSD-II Using Fully Deleted Adenovirus Based Vectors Expressing hGAA

Farber disease is a rare, lysosomal storage disorder that is caused by the deficient activity of the enzyme acid ceramidase. As a result, the sphingolipid ceramide accumulates in the lysosomal compartment. Patients typically present with subcutaneous granulomas, painful swollen joints and progressive neurological deterioration. Affected tissues show massive infiltrations of granulomatous cells and lipid-laden macrophages. Current treatment is only palliative and patients usually succumb to the disease as infants. While bone marrow transplantation has been shown to relieve some symptoms of the disease, it is does not provide a lasting cure since it is unsuccessful in resolving the neurological effects. Previously, our lab demonstrated the feasibility of gene therapy to correct Farber disease using an early recombinant onco-retroviral vector. Here we have developed novel recombinant onco-retroviral and lentiviral vectors to improve correction. A bi-cistronic onco-retroviral vector has been constructed that encodes human acid ceramidase and huCD25, a cell surface marker that allows for enrichment of transduced cells. This vector uses the MoMLV backbone and has been pseudotyped with the RD114 envelope. The lentiviral vector construct is a second generation, self-inactivating HIV-1-derived vector pseudotyped with the VSV-g envelope. Immortalized Farber patient fibroblasts and B cells have been transduced with both recombinant therapeutic viruses and subsequent quantitation of ceramide levels by thin layer chromatography showed that Farber patient cells were enzymatically corrected – while Farber fibroblasts contain 84% ceramide (as a percentage of sphingomyelin metabolites), normal fibroblasts, and Farber fibroblasts transduced with onco-retrovirus and with lentivirus show ceramide levels of 26%, 11% and 16%, respectively. Normal B cells, Farber B cells and Farber B cells transduced with the onco-retrovirus show ceramide levels of 11%, 76% and 16%, respectively. In addition, transduced fibroblasts show the ability to cross-correct non-transduced Farber fibroblasts, i.e. culture media from Farber fibroblasts transduced with onco-retrovirus and lentivirus were able to reduce ceramide levels in non-transduced Farber fibroblasts from 80% to 19% and 21%, respectively. Subsequently, the outcome of transplantation of transduced cord blood-derived hematopoietic stem/progenitor cells will be tested in vivo in a NOD/SCID mouse model. Late experiments using these novel vectors will also begin to address some of the central nervous system manifestations of the disease. These studies represent significant advances towards the development and clinical implementation of gene therapy for Farber disease as well as adding support to the concept that this class of disorders (lysosomal storage disorders) may be especially amenable to gene therapy.

Targeted adenoviral vectors

The practical implementation of gene therapy in the clinical setting mandates gene delivery vehicles, or vectors, capable of efficient gene delivery selectively to the target disease cells. The utility of adenoviral vectors for gene therapy is restricted by their dependence on the native adenoviral primary cellular receptor for cell entry. Therefore, a number of strategies have been developed to allow CAR-independent infection of specific cell types, including the use of bispecific conjugates and genetic modifications to the adenoviral capsid proteins, in particular the fibre protein. These targeted adenoviral vectors have demonstrated efficient gene transfer in vitro, correlating with a therapeutic benefit in preclinical animal models. Such vectors are predicted to possess enhanced efficacy in human clinical studies, although anatomical barriers to their use must be circumvented.

Oligonucleotide-mediated modulation of mammalian gene expression.

The notion that oligonucleotides can modulate gene-specific expression was established more than a decade ago. Recent advances in molecular genetics have broadened the armamentarium used to manipulate gene expression in biological systems including triplex DNA, antisense RNA/DNA, and ribozymes (catalytic RNA). These oligonucleotides demonstrated important early application to the elucidation of cellular signaling pathways. More recently, studies with these agents have probed their utility as potential therapeutic agents, especially in the realm of cancer. With the implementation of gene therapy in early clinical trials, oligonucleotide-mediated suppression of gene expression has emerged as an important strategy for gene therapy. This review will discuss the current knowledge in this field, focusing on the biology of triplex DNA, antisense oligonucleotides, and ribozymes.