Field Of Gene Therapy Research Articles

Gene Therapy Applications of Non-Human Lentiviral Vectors.

Recent commercialization of lentiviral vector (LV)-based cell therapies and successful reports of clinical studies have demonstrated the untapped potential of LVs to treat diseases and benefit patients. LVs hold notable and inherent advantages over other gene transfer agents based on their ability to transduce non-dividing cells, permanently transform target cell genome, and allow stable, long-term transgene expression. LV systems based on non-human lentiviruses are attractive alternatives to conventional HIV-1-based LVs due to their lack of pathogenicity in humans. This article reviews non-human lentiviruses and highlights their unique characteristics regarding virology and molecular biology. The LV systems developed based on these lentiviruses, as well as their successes and shortcomings, are also discussed. As the field of gene therapy is advancing rapidly, the use of LVs uncovers further challenges and possibilities. Advances in virology and an improved understanding of lentiviral biology will aid in the creation of recombinant viral vector variants suitable for translational applications from a variety of lentiviruses.

Clinical Adoption of Advanced Therapies: Challenges and Opportunities.

As the cell and gene therapy field matures the powerful therapeutic potential of these innovative therapies is starting to be shown, particularly in the fields of oncology and childhood immune deficiency diseases. However, as more therapies enter late stage clinical trials, advances and innovation are required in manufacturing, logistics, regulation, reimbursement and the healthcare setting to ensure that systems are in place to support wider clinical adoption of these promising treatments. A window of opportunity exists to implement new methodologies for best practice in both the ability to manufacture products reproducibly at scale, as well as ensuring healthcare systems are not overwhelmed by the variety and complexity of these new therapies and the additional burden they will place on already stretched facilities. If all interested parties work together it will be possible for the sector to develop the necessary processes, skilled staff and infrastructure needed as more treatments move from clinical trial to marketed products.

Overhauling CAR T Cells to Improve Efficacy, Safety and Cost.

Gene therapy is now surpassing 30 years of clinical experience and in that time a variety of approaches has been applied for the treatment of a wide range of pathologies. While the promise of gene therapy was over-stated in the 1990’s, the following decades were met with polar extremes between demonstrable success and devastating setbacks. Currently, the field of gene therapy is enjoying the rewards of overcoming the hurdles that come with turning new ideas into safe and reliable treatments, including for cancer. Among these modalities, the modification of T cells with chimeric antigen receptors (CAR-T cells) has met with clear success and holds great promise for the future treatment of cancer. We detail a series of considerations for the improvement of the CAR-T cell approach, including the design of the CAR, routes of gene transfer, introduction of CARs in natural killer and other cell types, combining the CAR approach with checkpoint blockade or oncolytic viruses, improving pre-clinical models as well as means for reducing cost and, thus, making this technology more widely available. While CAR-T cells serve as a prime example of translating novel ideas into effective treatments, certainly the lessons learned will serve to accelerate the current and future development of gene therapy drugs.

The Development and Application of a Base Editor in Biomedicine.

Using a base editor to generate monogenic disease models and correct pathogenic point mutations is a breakthrough technology for exploration and treatment of human diseases. As a burgeoning approach for genomic modification, the fused CRISPR/Cas9 with various deaminase separately has significantly increased the efficiency of producing a precise point mutation with minimal insertions or deletions (indels). Along with the flexibility and efficiency, a base editor has been widely used in many fields. This review discusses the recent development of a base editor, including evolution and advance, and highlights the applications and challenges in the field of gene therapy. Depending on rapid improvement and optimization of gene editing technology, the prospect of base editor is immeasurable.

Optimization of the quality by design approach for gene therapy products: A case study for adeno-associated viral vectors

The development of gene therapy products has been expanding globally, and among them, the recombinant adeno-associated virus (rAAV) vector is one of the most promising vectors for gene transfer. For efficient and rapid development of the manufacturing process and quality control strategy, the quality by design (QbD) approach can be as effective for gene therapy products as it is for gene recombinant proteins, which have been developed for decades. However, prior available knowledge required for the QbD approach is limited in the field of gene therapy. Here, we comprehensively review rAAV study results that can form the basis of QbD-based development and propose a critical quality attribute identification method suitable for gene therapy development. As a case study for rAAV, we propose a series of practical development steps, including a quality target product profile (QTPP) setting, identification of critical quality attributes (CQAs), repetitive risk assessment associated with process optimization, design space (DS) establishment, and control strategy using the QbD method. Our case study, which was based on publicly available literature, is a basic model that can be augmented by unique data pertaining to specific products. An improvement in rAAV development is expected using this model as the first step.

Clinical trials in the era of Covid-19: successes, failures & ongoing challenges

In our July issue, Sven Kili focuses his regular analysis of current clinical trends on the far-reaching and in some cases devastating impact of the Covid-19 pandemic. From new investment trends and delayed trials to changes to approval processes, the shockwaves of the pandemic have been felt in almost every corner of the cell and gene therapy field.

Physicochemical characterization and targeting performance of triphenylphosphonium nano-polyplexes

Mitochondrial gene therapy can be seen as a promising tool and a revolutionary approach towards mitochondrial diseases arising from mitochondrial DNA mutations. To pursue this goal, the mitochondria-targeted polycation polyethylenimine (PEI)/triphenylphosphonium (TPP) conjugate has been used to complex and condense both GFP or ND1 encoded plasmid DNA (pDNA) aiming at the formation of suitable vectors for mitochondrial gene therapy. The properties of pDNA based TPP-polyplexes have been addressed and appear to be valuable for gene delivery applications. In vitro studies using fluorescent pDNA demonstrated the internalization of the developed carriers and mitochondria targeting. Evidences of gene expression into mitochondria are also reported. Following this achievement, significant levels of both mitochondrial recoded GFP and ND1 proteins were quantified. This work represents a great step towards effective mitochondria-targeting gene delivery and functional protein expression, providing a significant contribution in mitochondrial gene therapy field.

Tropism-facilitated delivery of CRISPR/Cas9 system with chimeric antigen receptor-extracellular vesicles against B-cell malignancies

The CRISPR/Cas9 system is an efficient genome-editing system that has been successfully applied in the field of gene therapy. However, clinical applications of the CRISPR/Cas9 system are limited by the delivery method and safety concerns. Extracellular Vesicles (EVs) can be released from almost every type of cell, and they act as shuttles to convey molecules between cells. Here, we used EVs derived from epithelial cells as a biosafety delivery platform for the CRISPR/Cas9 system and modified the EVs with a chimeric-antigen receptor (CAR) to give them selective tropism to tumors. Compared to normal EVs, CAR-EVs accumulated in cancer tumors rapidly and released the CRISPR/Cas9 system targeting the MYC oncogene efficiently, both in vitro and in vivo. Taken together, the combination of EV and CAR was confirmed to be a novel strategy facilitating the use of natural gene therapy platforms in cancer treatment in this proof-of-concept research.

Gene therapy development in hearing research in China.

Sensorineural hearing loss, the most common form of hearing impairment, is mainly attributable to genetic mutations or acquired factors, such as aging, noise exposure, and ototoxic drugs. In the field of gene therapy, advances in genetic and physiological studies and profound increases in knowledge regarding the underlying mechanisms have yielded great progress in terms of restoring the auditory function in animal models of deafness. Nonetheless, many challenges associated with the translation from basic research to clinical therapies remain to be overcome before a total restoration of auditory function can be expected. In recent years, Chinese research teams have promoted various developmental efforts in this field, including gene sequencing to identify additional potential loci that cause deafness, studies to elucidate the underlying molecular mechanisms, and research to optimize vectors and delivery routes. In this review, we summarize the state of the field and focus mainly on the progress of gene therapy in animal model studies and the optimization of therapeutic strategies in China.

AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity

The gene therapy field has been galvanized by two technologies that have revolutionized treating genetic diseases: vectors based on adeno-associated viruses (AAVs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene-editing tools. When combined into one platform, these safe and broadly tropic biotherapies can be engineered to target any region in the human genome to correct genetic flaws. Unfortunately, few investigations into the design compatibility of CRISPR components in AAV vectors exist. Using AAV-genome population sequencing (AAV-GPseq), we previously found that self-complementary AAV vector designs with strong DNA secondary structures can cause a high degree of truncation events, impacting production and vector efficacy. We hypothesized that the single-guide RNA (sgRNA) scaffold, which contains several loop regions, may also compromise vector integrity. We have therefore advanced the AAV-GPseq method to also interrogate single-strand AAV vectors to investigate whether vector genomes carrying Cas9-sgRNA cassettes can cause truncation events. We found that on their own, sgRNA sequences do not produce a high degree of truncation events. However, we demonstrate that vector genome designs that carry dual sgRNA expression cassettes in tail-to-tail configurations lead to truncations. In addition, we revealed that heterogeneity in inverted terminal repeat sequences in the form of regional deletions inherent to certain AAV vector plasmids can be interrogated.

Safeguards for Using Viral Vector Systems in Human Gene Therapy: A Resource for Biosafety Professionals Mitigating Risks in Health Care Settings.

Health care workers who work daily with human body fluids and hazardous drugs are among those at the highest risk of occupational exposure to these agents. The Occupational Safety and Health Administration's (OSHA) Bloodborne Pathogens Standard (29 CFR 1910.1030) prescribes safeguards to protect workers against health hazards related to bloodborne pathogens. Similarly, the United States Pharmacopeia General Chapter 800 (USP <800>), a standard first published in February 2016 and implementation required by December 2019, addresses the occupational exposure risks of health care workers at organizations working with hazardous drugs. With emerging technologies in the field of gene therapy, these occupational exposure risks to health care workers now extend beyond those associated with bloodborne pathogens and hazardous drugs and now include recombinant DNA. The fifth edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) and the National Institutes of Health Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) mostly govern work with biohazardous agents and recombinant DNA in a laboratory research setting. When gene therapy products are utilized in a hospital environment, health care workers have very few resources to identify and reduce the risks associated with product use during and after the administration of treatments. At the Abigail Wexner Research Institute at Nationwide Children's Hospital, a comprehensive gap analysis was executed between the research and health care environment to develop a program for risk mitigation. The BMBL, NIH Guidelines, World Health Organization Biosafety Manual, OSHA Bloodborne Pathogens Standard, and USP <800> were used to develop a framework for the gap analysis process. The standards and guidelines for working with viral vector systems in a research laboratory environment were adapted to develop a program that will mitigate the risks to health care workers involved in the preparation, transportation, and administration of gene therapies as well as subsequent patient care activities. The gap analysis identified significant differences in technical language used in daily operations, work environment, training and education, disinfection practices, and policy development between research and health care settings. These differences informed decisions and helped the organization develop a collaborative framework for risk mitigation when a gene therapy product enters the health care setting. With continuing advances in the field of gene therapy, the oversight structure needs to evolve for the health care setting. To deliver the best outcomes to the patients of these therapies, researchers, Institutional Biosafety Committees, and health care workers need to collaborate on training programs to safeguard the public trust in the use of this technology both in clinical trials and as FDA-approved therapeutics.

Role of Lipid-Based and Polymer-Based Non-Viral Vectors in Nucleic Acid Delivery for Next-Generation Gene Therapy.

The field of gene therapy has experienced an insurgence of attention for its widespread ability to regulate gene expression by targeting genomic DNA, messenger RNA, microRNA, and short-interfering RNA for treating malignant and non-malignant disorders. Numerous nucleic acid analogs have been developed to target coding or non-coding sequences of the human genome for gene regulation. However, broader clinical applications of nucleic acid analogs have been limited due to their poor cell or organ-specific delivery. To resolve these issues, non-viral vectors based on nanoparticles, liposomes, and polyplexes have been developed to date. This review is centered on non-viral vectors mainly comprising of cationic lipids and polymers for nucleic acid-based delivery for numerous gene therapy-based applications.

305 In vivo correction of recessive dystrophic epidermolysis bullosa (RDEB) by direct cutaneous COL7A1 gene replacement: Results of a phase 1-2 trial

The use of direct gene transfer to correct genetic skin disease has been a longstanding goal in the gene therapy field. Here we demonstrate correction of the genetic disorder RDEB by the application of a replication defective herpes simplex virus-1 vector encoding the type VII collagen (C7) gene COL7A1, bercolagene telserpavec (BVEC), directly to skin via a phase 1-2 clinical trial. This trial was an intra-patient comparison of recurrent and chronic wounds administered with either topical BVEC or topical placebo applied every other day until healing.

Selection of Water-Soluble Chitosan by Microwave-Assisted Degradation and pH-Controlled Precipitation.

In the field of gene therapy, chitosan (CS) gained interest for its promise as a non-viral DNA vector. However, commercial sources of CS lack precise characterization and do not generally reach sufficient solubility in aqueous media for in vitro and in vivo evaluation. As low molecular weight CS showed improved solubility, we investigated the process of CS depolymerization by acidic hydrolysis, using either long time heating at 80 °C or short time microwave-enhanced heating. The resulting depolymerized chitosan (dCS) were analyzed by gel permeation chromatography (GPC) and 1H nuclear magnetic resonance (NMR) to determine their average molecular weight (Mn, Mp and Mw), polydispersity index (PD) and degree of deacetylation (DD). We emphasized the production of water-soluble CS (solubility > 5 mg/mL), obtained in reproducible yield and characteristics, and suitable for downstream functionalization. Optimal microwave-assisted conditions provided dCS with a molecular weight (MW) = 12.6 ± 0.6 kDa, PD = 1.41 ± 0.05 and DD = 85%. While almost never discussed in the literature, we observed the partial post-production aggregation of dCS when exposed to phase changes (from liquid to solid). Repeated cycles of freezing/thawing allowed the selection of dCS fractions which were exempt of crystalline particles formation upon solubilization from frozen samples.

Application of quantitative and engineering biology in mRNA gene therapy

<p indent=0mm>As a transient vector of gene information, messenger RNA (mRNA) is a multifunctional, flexible and safe gene therapy. However, due to the general instability and immunogenicity of mRNA molecules, mRNA gene therapy has not been popularized at first. Over the past few decades, the cross-integration of quantitative and engineering biology has led to a gradual shift from the concept validation phase to the clinical treatment phase. In this paper, we discussed the application of quantitative and engineering biology in mRNA gene therapy from the functional structure of mRNA, design and technical innovation of <italic>in vitro</italic> synthetic mRNA. Effective engineering approaches will help to establish a precise mRNA gene therapy platform for a variety of indications and genetic backgrounds. Although mRNA therapy is still in the pre-clinical phase in most indications, the accumulation of various engineering and quantitative bio-design methods may broaden the path for next-generation mRNA gene therapy. mRNA is an intermediate of genetic information and a template for the biological expression of proteins. Therefore, exogenous mRNA can be introduced into target cells to express the protein of interest. In the early 1970s, Gurdon first verified the above concept in <italic>Xenopus oocytes</italic> using microinjection. The advantages of mRNA-based gene therapy methods are: First, mRNA therapy has a broader application. mRNA does not need to enter the nucleus to express proteins in the cytoplasm to achieve its function. Therefore, mRNA can be played in cells that divide slowly or do not divide role. Second, mRNA therapy has higher safety, due to its transient expression and the extremely low possibility of genome integration, thereby reducing the two main risks associated with gene therapy (host genome integration and mutations in key regions). Third, the half-life and expression abundance of mRNA synthesized <italic>in vitro</italic> can be determined by the structure and component design, so that the pharmacokinetics of mRNA drugs can be better controlled. This has created favorable conditions for the pharmaceutical good manufacturing practice (GMP) of mRNA synthetic drugs. In addition, therapeutic proteins synthesized using the natural mechanism of cells have natural post-translational modifications and suitable protein folding effects, which are more advantageous than recombinant proteins synthesized <italic>in vitro</italic>. In recent years, mRNA, as a therapeutic method, has received increasing attention in the field of gene therapy. Currently, <italic>in vitro</italic>-transcribed (IVT) mRNA has been used in pre-clinical and clinical trials, including cancer treatment, vaccine development, and protein replacement drug development. Compared with cancer and vaccine treatment, most protein replacement therapies using mRNA are still in preclinical development stage.

Research Advances on Ex Vivo Expansion Strategy of Hematopoietic Stem Cells--Review

Abstract Hematopoietic stem cells (HSCs), as a kind of adult stem cell, is first used in clinical practice. It has been widely used in hematopoietic stem cell transplantation and has broad application prospects in the field of gene therapy. However, the shortage of HSC is still the main bottleneck restricting the clinical application of HSC transplantation, especially cord blood HSC transplantation. With the continuous improvement of the concept of HSC and the in-depth study of the molecular mechanism regulating HSC self-renewal and differentiation, it have been explored that a variety of strategies for ex vivo expansion of HSC, such as adding small molecule compounds in vitro culture system, simulating bone marrow microenvironment, regulating HSC metabolism and biomaterial-based amplification methods. In this review, recent advances in research of ex vivo expansion strategy of HSC are summarized and discussed.

Cancer Gene Therapy and its techniques in Cancer Biology

The researches conducted over the past two decades, in the field of human genomics has indicated that cancer in the genome is caused by somatic aberration. This discovery has sparked excitement in cancer researchers. Most people are now using genetic engineering therapeutic methods for advance malignancy treatment or discover a possible therapy of this infection. The field of gene therapy provides a range of groundbreaking therapies which can be significant for the prevention of deaths from cancer. In immunotherapy, genetically engineered cell and virus vectors are typically used to stimulate the immune system and destroy the infected cells. Recent trials of the new generation vaccines with a broad range of cancers have shown encouraging results. Oncolytic virotherapy which is a new method of treatment that shows a great deal of scope and chance, especially with metastatic cancers that uses viral vectors that repeat cancer-infected cells to cause cell death. A new approach to treatment by inserting fresh gene into tumorous cell tissues is the gene transfer technique that causes cell death or reduces cancer growing. Such therapies may be used as a standalone therapy or in conjunction with present treatments as they develop to make cancer a curable disease. It is expected to show a significant part in potential tumor treatment with other types of malignancy treatment, like radiation therapy, surgery and chemotherapy, as part of a multimodality procedure.

Gene Therapy for Cystic Fibrosis: Progress and Challenges of Genome Editing.

Since the early days of its conceptualization and application, human gene transfer held the promise of a permanent solution to genetic diseases including cystic fibrosis (CF). This field went through alternated periods of enthusiasm and distrust. The development of refined technologies allowing site specific modification with programmable nucleases highly revived the gene therapy field. CRISPR nucleases and derived technologies tremendously facilitate genome manipulation offering diversified strategies to reverse mutations. Here we discuss the advancement of gene therapy, from therapeutic nucleic acids to genome editing techniques, designed to reverse genetic defects in CF. We provide a roadmap through technologies and strategies tailored to correct different types of mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, and their applications for the development of experimental models valuable for the advancement of CF therapies.

Bio-inspired gene carriers with low cytotoxicity constructed via the assembly of dextran nanogels and nano-coacervates.

Aim: To achieve safe and biocompatible gene carriers. Materials & methods: A core/shell-structured hierarchical carrier with an internal peptide/gene coacervate 'core' and a dextran nanogel 'shell' on the surface has been designed. Results: The dextran nanogels shield coacervate (DNSC) can effectively condense genes and release them in reducing environments. The dextran nanogel-based 'shell' can effectively shield the positive charge of the peptide/gene coacervate 'core', thus reducing the side effects of cationic gene carriers. In contrast with the common nonviral gene carriers that had high cytotoxicities, the DNSC showed a high transfection efficiency while maintaining a low cytotoxicity. Conclusion: The DNSC provides an effective environmentally responsive gene carrier with potential applications in the fields of gene therapy and gene carrier development.

High-Capacity Adenoviral Vectors: Expanding the Scope of Gene Therapy.

The adaptation of adenoviruses as gene delivery tools has resulted in the development of high-capacity adenoviral vectors (HC-AdVs), also known, helper-dependent or “gutless”. Compared with earlier generations (E1/E3-deleted vectors), HC-AdVs retain relevant features such as genetic stability, remarkable efficacy of in vivo transduction, and production at high titers. More importantly, the lack of viral coding sequences in the genomes of HC-AdVs extends the cloning capacity up to 37 Kb, and allows long-term episomal persistence of transgenes in non-dividing cells. These properties open a wide repertoire of therapeutic opportunities in the fields of gene supplementation and gene correction, which have been explored at the preclinical level over the past two decades. During this time, production methods have been optimized to obtain the yield, purity, and reliability required for clinical implementation. Better understanding of inflammatory responses and the implementation of methods to control them have increased the safety of these vectors. We will review the most significant achievements that are turning an interesting research tool into a sound vector platform, which could contribute to overcome current limitations in the gene therapy field.