Field Of Gene Therapy Research Articles

Standardization & analytical tool innovation in the cell & gene therapy field

David McCall, Editor, Cell and Gene Therapy Insights, speaks to Fouad Atouf, Vice President, Global Biologics, United States PharmacopeiaFouad Atouf is Vice President, Global Biologics, for USP. He leads all scientific activities related to the development and maintenance of documentary and reference standards for biologics and advanced therapies as well as the oversees biologics laboratories in USP–US and USP–India. Dr Atouf has been at USP for over 15 years and served in a variety of scientific leadership roles including being the regional champion for the Middle East and North Africa Region, where he helped facilitate, programs designed to enhance the understanding of the role of regulations and standards in the registration of medicinal products. Dr Atouf has a strong background and experience in the development and standardization of cellular and tissue-based products. Prior to joining USP in 2006, his research at the US National Institutes of Health focused on developing methods for the in vitro generation of cell-based therapies for diabetes. Dr Atouf is the author of numerous publications in peer-reviewed journals and a frequent speaker at national and international scientific conferences. Dr Atouf earned his Master’s degree in Biochemistry and his PhD in Cell Biology from the Pierre & Marie Curie University, Paris, France.

Analytical development insights spanning viral vectors & gene editing

David McCall, Commissioning Editor of Cell & Gene Therapy Insight speaks to Santoshkumar Khatwani, Director Analytical Development, Sangamo Therapeutics, Inc. Dr Santosh Khatwani graduated from the University of Kentucky in 2010 with PhD in Chemistry under the guidance of Prof. Sylvia Daunert. The research work focused on the use of generically engineered recombinant protein and enzymes for bio sensing and biomaterial applications. Furthermore, he obtained postdoctoral training at the University of Minnesota (until 2012) under the supervision of Prof T Andrew Taton and Prof Mark D Distefano with focus on the use of site-specific covalent conjugation for synthesis of protein–DNA conjugates and analytical assays for the generating novel engineered enzymes with improved functional attributes. Dr Khatwani then joined BioVision Inc. in 2012 and served under different capacities until 2017 where he oversaw the manufacture, testing and release of several recombinant protein, enzyme and assays for various metabolically important enzymes. Furthermore, Dr. Khatwani joined the field of cell and gene therapy at multiple companies including Thermofisher Viral Vector Services (formerly Brammer Bio, 2017–2018), Asklepios Biopharmaceuticals (2019) and Sangamo Therapeutics (2018–2019, 2019-present) at various leadership roles where he has led the development of various analytical assays including physicochemical, biophysical and biological assays to support the manufacture and release of viral vectors in cell and gene therapy. Currently Dr. Khatwani is serving as Director of Analytical Development at Sangamo Therapeutics with strong focus on developing analytical solutions and CMC in support of product development at early and late phase of the clinical development.

Reflections on a life in gene therapy

David McCall, Editor, Cell and Gene Therapy Insights speaks to Ronald G Crystal, Professor and Chairman of the Department of Genetic Medicine at Weill Cornell Medical CollegeRonald G Crystal, MD, is Professor and Chairman of the Department of Genetic Medicine at Weill Cornell Medical College, where he is also Bruce Webster Professor of Internal Medicine and Director of the Belfer Gene Therapy Core Facility. After earning a BA in physics from Tufts University and an MS in physics and an MD from the University of Pennsylvania, Dr Crystal served as Chief of the Pulmonary Branch of the National Heart, Lung and Blood Institute. His initial research at Weill Cornell focused on inflammatory diseases of the lung, shifting in the late 1980s to the field of gene therapy in which Dr Crystal became a pioneer. Recently developed gene therapy programs include the treatment of Friedreich’s ataxia, cocaine addiction and Alzheimer’s disease.

Roundtable Roundup: strategies for scaling up and out in gene therapy manufacturing - addressing AAV’s growing pains

In a recent Expert Roundtable discussion, we spoke to three experts about the challenges the rapidly expanding gene therapy field is facing in scaling up adeno-associated vector manufacturing, and the potential solutions. Here, we sum up some of their key thoughts.

Adeno-Associated Viral Vectors as Versatile Tools for Neurological Disorders: Focus on Delivery Routes and Therapeutic Perspectives.

It is without doubt that the gene therapy field is currently in the spotlight for the development of new therapeutics targeting unmet medical needs. Thus, considering the gene therapy scenario, neurological diseases in general and neurodegenerative disorders in particular are emerging as the most appealing choices for new therapeutic arrivals intended to slow down, stop, or even revert the natural progressive course that characterizes most of these devastating neurodegenerative processes. Since an extensive coverage of all available literature is not feasible in practical terms, here emphasis was made in providing some advice to beginners in the field with a narrow focus on elucidating the best delivery route available for fulfilling any given AAV-based therapeutic approach. Furthermore, it is worth nothing that the number of ongoing clinical trials is increasing at a breath-taking speed. Accordingly, a landscape view of preclinical and clinical initiatives is also provided here in an attempt to best illustrate what is ongoing in this quickly expanding field.

The Learning Curve of Murine Subretinal Injection Among Clinically Trained Ophthalmic Surgeons.

PurposeSubretinal injection (SRI) in mice is widely used in retinal research, yet the learning curve (LC) of this surgically challenging technique is unknown.MethodsTo evaluate the LC for SRI in a murine model, we analyzed training data from three clinically trained ophthalmic surgeons from 2018 to 2020. Successful SRI was defined as either the absence of retinal pigment epithelium (RPE) degeneration after phosphate buffered saline injection or the presence of RPE degeneration after Alu RNA injection. Multivariable survival-time regression models were used to evaluate the association between surgeon experience and success rate, with adjustment for injection agents, and to calculate an approximate case number to achieve a 95% success rate. Cumulative sum (CUSUM) analyses were performed and plotted individually to monitor each surgeon's simultaneous performance.ResultsDespite prior microsurgery experience, the combined average success rate of the first 50 cases in mice was only 27%. The predicted SRI success rate did not reach a plateau above 95% until approximately 364 prior cases. Using the 364 training cases as a cutoff point, the predicted probability of success for cases 1 to 364 was 65.38%, and for cases 365 to 455 it was 99.32% (P < 0.0001). CUSUM analysis showed an initial upward slope and then remained within the decision intervals with an acceptable success rate set at 95% in the late stage.ConclusionsThis study demonstrates the complexity and substantial LC for successful SRI in mice with high confidence. A systematic training system could improve the reliability and reproducibility of SRI-related experiments and improve the interpretation of experimental results using this technique.Translational RelevanceOur prediction model and monitor system allow objective quantification of technical proficiency in the field of subretinal drug delivery and gene therapy for the first time, to the best of our knowledge.

Nonviral Delivery Systems of mRNA Vaccines for Cancer Gene Therapy.

In recent years, the use of messenger RNA (mRNA) in the fields of gene therapy, immunotherapy, and stem cell biomedicine has received extensive attention. With the development of scientific technology, mRNA applications for tumor treatment have matured. Since the SARS-CoV-2 infection outbreak in 2019, the development of engineered mRNA and mRNA vaccines has accelerated rapidly. mRNA is easy to produce, scalable, modifiable, and not integrated into the host genome, showing tremendous potential for cancer gene therapy and immunotherapy when used in combination with traditional strategies. The core mechanism of mRNA therapy is vehicle-based delivery of in vitro transcribed mRNA (IVT mRNA), which is large, negatively charged, and easily degradable, into the cytoplasm and subsequent expression of the corresponding proteins. However, effectively delivering mRNA into cells and successfully activating the immune response are the keys to the clinical transformation of mRNA therapy. In this review, we focus on nonviral nanodelivery systems of mRNA vaccines used for cancer gene therapy and immunotherapy.

Intrauterine Fetal Gene Therapy: Is That the Future and Is That Future Now?

Researchers are looking into techniques to intervene sooner and earlier in the disease process thanks to advances in disease genetics, etiologies, and prenatal diagnosis. We conducted a literature search in PubMed-indexed journals to provide an overview of the evolution of gene therapy, rationale for prenatal gene therapy, uses and risks of gene therapy, and ethical issues following the usage of gene therapy. Recent animal research has revealed that transmitting genetic material to a growing fetus through viral and non-viral vectors is conceivable besides proving how gene-editing technology is achieved by various mechanisms that utilize zinc finger nucleases, TAL effector nucleases, and clustered short palindromic repeats-Cas9 complex. This review offers an overview of the current knowledge in the field of prenatal gene therapy, as well as potential future research avenues. In addition, it weighs the risks of prenatal gene therapy, such as oncogenesis, genetic mutation transfer from mother to child, and fetal disruption, against the expected benefits, such as preventing the development of severe early-onset illness symptoms, targeting previously inaccessible organs, and establishing tolerance to the therapeutic transgenic protein, all of which lead to permanent somatic gene correction.This review discusses the scientific, ethical, legal, and sociological implications of these groundbreaking genetic disease prevention techniques, as well as the parameters that must be satisfied for a future clinical application to be considered.

Regenerative medicine: new paradigms

<div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://bioinsights.blob.core.windows.net/uploads/1c0c05c2e29e4517a0c787cd83475dc2.png" data-image="1"></div><div class="author-det"> <i>“There is a lot to look forward to in the cell and gene therapy field. But there are a few key challenges that ARM and the sector are addressing.”</i> </div></div></div>

Advancing engineered cell &amp; gene therapy with precision gene editing

The increased range and accessibility of gene modification technologies has had a great impact on the cell and gene therapy field, with CRISPR-Cas9 in particular garnering increased attention in the last decade. But is it always the best tool for the job? In this article, CRISPR and base editing techniques will be compared – demonstrating the key advantages base editing technology can offer to the field in terms of safety and efficiency, and how increasingly precise gene editing approaches can help to deliver the next generation of advanced therapies.

HIV-2/SIV Vpx antagonises NF-\u03baB activation by targeting p65

BackgroundThe NF-κB family of transcription factors and associated signalling pathways are abundant and ubiquitous in human immune responses. Activation of NF-κB transcription factors by viral pathogen-associated molecular patterns, such as viral RNA and DNA, is fundamental to anti-viral innate immune defences and pro-inflammatory cytokine production that steers adaptive immune responses. Diverse non-viral stimuli, such as lipopolysaccharide and cytokines, also activate NF-κB and the same anti-pathogen gene networks. Viruses adapted to human cells often encode multiple proteins targeting the NF-κB pathway to mitigate the anti-viral effects of NF-κB-dependent host immunity.ResultsIn this study we have demonstrated using a variety of assays, in a number of different cell types including primary cells, that plasmid-encoded or virus-delivered simian immunodeficiency virus (SIV) accessory protein Vpx is a broad antagonist of NF-κB signalling active against diverse innate NF-κB agonists. Using targeted Vpx mutagenesis, we showed that this novel Vpx phenotype is independent of known Vpx cofactor DCAF1 and other cellular binding partners, including SAMHD1, STING and the HUSH complex. We found that Vpx co-immunoprecipitated with canonical NF-κB transcription factor p65, but not NF-κB family members p50 or p100, preventing nuclear translocation of p65. We found that broad antagonism of NF-κB activation by Vpx was conserved across distantly related lentiviruses as well as for Vpr from SIV Mona monkey (SIVmon), which has Vpx-like SAMHD1-degradation activity.ConclusionsWe have discovered a novel mechanism by which lentiviruses antagonise NF-κB activation by targeting p65. These findings extend our knowledge of how lentiviruses manipulate universal regulators of immunity to avoid the anti-viral sequelae of pro-inflammatory gene expression stimulated by both viral and extra-viral agonists. Importantly our findings are also relevant to the gene therapy field where virus-like particle associated Vpx is routinely used to enhance vector transduction through antagonism of SAMHD1, and perhaps also through manipulation of NF-κB.Graphical

Newer therapeutic agents for retinal diseases

ABSTRACT Introduction Last few decades in medical retina have encountered significant development in the field of pharmacotherapy. With better understanding of molecular mechanisms and disease pathophysiology, alternate pathways are being targeted in various clinical trials. Areas covered In this review, a comprehensive list of newer therapeutic agents in various retinal conditions like diabetic macular edema (DME), age-related macular degeneration (ARMD), inherited retinal diseases (IRDs) and non-arteritic ischemic optic neuropathy (NAION) have been elucidated. The review highlights alternate pathways involved in pathogenesis of DME and ARMD, and gives a brief summary of several phase I and phase II trials targeting these pathways. The review also briefly highlights the advancements in the field of gene therapy, stem cell transplantation, neuro-protection and retinal implants in IRDs. Lastly, available treatment options for NAION have been discussed. Expert opinion Evidence based medicine has allowed researches and vision scientists to explore newer drugs and target alternate disease pathways. Promising results with initial clinical trials will pave the pathway for larger studies and allow clinicians to overcome treatment burden presently associated with available treatment options.

Gene Therapy Strategies Targeting Aging-Related Diseases.

Rapid advancements have taken place in gene therapy technology. However, effective methods for treating aging- or age-related chronic diseases, which are often closely related to genes or even multiple genes, are still lacking. The path to developing cures is winding, while gene therapy that targets genes related to aging represents an exciting research direction with tremendous potential. Among aging-related genes, some candidates have been studied at different levels, from cell to organismal levels (e.g., mammalian models) with different methods, from overexpression to gene editing. The TERT and APOE have even entered the stage of clinical trials. Even those displaying only a preliminary association with diseases have potential applications. This article discusses the foundations and recent breakthroughs in the field of gene therapy, providing a summary of current mainstream strategies and gene therapy products with clinical and preclinical applications. Finally, we review representative target genes and their potential for treating aging or age-related diseases.

Gene therapy: from catastrophe to cure in 20 years

In 1999, the gene therapy field was rocked by the death of an 18-year-old clinical trial participant, Jesse Gelsinger. Gelsinger suffered from ornithine transcarbamylase deficiency (OTCD), an X-linked genetic disease resulting from a mutation in the OTC gene, which leads to a build up of ammonia in the liver and blood. Gelsinger was the 18th […]

Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of DNA-modifying enzymes may cause cytotoxic and genotoxic side effects and is therefore unwanted in therapeutic approaches. Consequently, development of new and potent short-term delivery methods is of utmost importance. Recently, we developed non-integrating gammaretrovirus- and MS2 bacteriophage-based Gag.MS2 (g.Gag.MS2) particles for transient transfer of non-retroviral CRISPR-Cas9 RNA into target cells. In the present study, we further improved the technique by transferring the system to the alpharetroviral vector platform (a.Gag.MS2), which significantly increased CRISPR-Cas9 delivery into target cells and allowed efficient targeted knockout of endogenous TP53/Trp53 genes in primary murine fibroblasts as well as primary human fibroblasts, hepatocytes, and cord-blood-derived CD34+ stem and progenitor cells. Strikingly, co-packaging of Cas9 mRNA and multiple single guide RNAs (sgRNAs) into a.Gag.MS2 chimera displayed efficient targeted knockout of up to three genes. Co-transfection of single-stranded DNA donor oligonucleotides during CRISPR-Cas9 particle production generated all-in-one particles, which mediated up to 12.5% of homology-directed repair in primary cell cultures. In summary, optimized a.Gag.MS2 particles represent a versatile tool for short-term delivery of DNA-modifying enzymes into a variety of target cells, including primary murine and human cells.

Cell-Based Determination of Neutralizing Antibodies Against Adeno-Associated Virus in Cardiac Gene Therapy.

The field of cardiac gene therapy has seen the rising use of adeno-associated viral (AAV) vectors as a promising therapeutic option for cardiac diseases and heart failure. To achieve intended results of AAV delivery, a majority of clinical studies screen patients for existing neutralizing antibodies that could inhibit the effects of the administered AAV and confound treatment efficacy. The cell-based neutralizing antibody assay offers a method of quantifying and identifying a patient's existing neutralizing antibodies against specific serotypes. Combined with the luciferase assay, the neutralizing antibody assay tests the ability of patient antibodies in the blood to prevent gene transduction of AAV-encoded luciferase gene at ranging serial dilutions. This chapter provides a protocol and experimental techniques to determine the presence of neutralizing antibodies against AAV in the blood.

Medical application of Chitosan

By using the deacetylation method, chitin is converted into bioproduct chitosan. Deacetylation can be accomplished using chemical or biological mechanisms. Due to its biocompatibility, nontoxicity, biodegradability, natural origin, and resemblance to human macromolecules, it is useful in medicine. Chitosan may have antibacterial and antioxidant properties. Additionally, it could be used in biotechnology, agriculture, gene therapy, food technology, medication delivery, cancer therapy, and other fields. The objective of the current review was to list the most significant applications of Chitosan in the biomedical field.

Development and Evaluation of Aceclofenac Liposomes

This review gives concise information about the application of dendrimers as drug delivery carrier in the field of drug delivery. Due to their unique architecture these have improved physical and chemical properties. Due to their terminal groups these show high solubility, miscibility and reactivity. Dendrimers have well defined size, shape, molecular weight and monodispersity. These properties make the dendrimers a suitable carrier in drug delivery application. Dendrimers are unimolecular miceller in nature and due to this enhances the solubility of poorly soluble drugs. Their compatibility with DNA, heparin and polyanions make them more versatile. Dendrimers, also referred as modern day polymers, they offer much more good properties than the conventional polymers. Due to their multivalent and mono disperse character dendrimers have stimulated wide interest in the field of chemistry biology, drug delivery, gene therapy and chemotherapy. Self-assembly produces a faster means of generating nanoscopic functional and structural systems. But their actual utility in drug delivery can be assessed only after deep understanding of factors affecting their properties and their behaviour in vivo. Keywords: Dendrimers, Drug targeting, nanoscale carriers.

Dendrimers as Drug Delivery Carriers in the Dentistry

This review gives concise information about the application of dendrimers as drug delivery carrier in the field of drug delivery. Due to their unique architecture these have improved physical and chemical properties. Due to their terminal groups these show high solubility, miscibility and reactivity. Dendrimers have well defined size, shape, molecular weight and monodispersity. These properties make the dendrimers a suitable carrier in drug delivery application. Dendrimers are unimolecular miceller in nature and due to this enhances the solubility of poorly soluble drugs. Their compatibility with DNA, heparin and polyanions make them more versatile. Dendrimers, also referred as modern day polymers, they offer much more good properties than the conventional polymers. Due to their multivalent and mono disperse character dendrimers have stimulated wide interest in the field of chemistry biology, drug delivery, gene therapy and chemotherapy. Self-assembly produces a faster means of generating nanoscopic functional and structural systems. But their actual utility in drug delivery can be assessed only after deep understanding of factors affecting their properties and their behaviour in vivo. Keywords: Dendrimers, Drug targeting, nanoscale carriers.

Application of CRISPR/Cas9 in Alzheimer's Disease.

Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disorder clinically characterized by cognitive impairment, abnormal behavior, and social deficits, which is intimately linked with excessive β-amyloid (Aβ) protein deposition along with many other misfolded proteins, neurofibrillary tangles formed by hyperphosphorylated tau protein aggregates, and mitochondrial damage in neurons, leading to neuron loss. Currently, research on the pathological mechanism of AD has been elucidated for decades, still no effective treatment for this complex disease was developed, and the existing therapeutic strategies are extremely erratic, thereby leading to irreversible and progressive cognitive decline in AD patients. Due to gradually mental dyscapacitating of AD patients, AD not only brings serious physical and psychological suffering to patients themselves, but also imposes huge economic burdens on family and society. Accordingly, it is very imperative to recapitulate the progress of gene editing-based precision medicine in the emerging fields. In this review, we will mainly focus on the application of CRISPR/Cas9 technique in the fields of AD research and gene therapy, and summarize the application of CRISPR/Cas9 in the aspects of AD model construction, screening of pathogenic genes, and target therapy. Finally, the development of delivery systems, which is a major challenge that hinders the clinical application of CRISPR/Cas9 technology will also be discussed.