Gene Therapy Applications Research Articles

Adeno-Associated Viral Vector Immobilization and Local Delivery from Bare Metal Surfaces.

Spatially and temporally controlled delivery of biologicals, including gene vectors, represents an unmet need for regenerative medicine and gene therapy applications. Here we describe a method of reversible attachment of serotype 2 adeno-associated viral vectors (AAV2) to metal surfaces. This technique enables localized delivery of the vector to the target cell population in vitro and in vivo with the subsequent effective transduction of cells adjacent to the metal substrate. The underlying bioengineering approach employs coordination chemistry between the bisphosphonic groups of polyallylamine bisphosphonates and the metal atoms on the surface of metallic samples. Formation of a stable polybisphosphonate monolayer with plentiful allyl-derived amines allows for further chemical modification to consecutively append thiol-modified protein G, an anti-AAV2 antibody, and AAV2 particles. Herein we present a detailed protocols for the metal substrate modification, for the visualization of the metal surface-immobilized vector using direct and indirect fluorescent AAV2 labeling and scanning electron microscopy, for quantification of the surface-immobilized vector load with RT-PCR, and for the localized vector transduction in vitro and in vivo.

Updates on Cardiac Gene Therapy Research and Methods: Overview of Cardiac Gene Therapy.

Gene therapy has made a significant progress in clinical translation over the past few years with several gene therapy products currently approved or anticipating approval for clinical use. Cardiac gene therapy lags behind that of other areas of diseases, with no application of cardiac gene therapy yet approved for clinical use. However, several clinical trials for gene therapy targeting the heart are underway, and innovative research studies are being conducted to close the gap. The second edition of Cardiac Gene Therapy in Methods in Molecular Biology provides protocols for cutting-edge methodologies used in these studies. In this chapter, we discuss recent updates on cardiac gene therapy studies and provide an overview of the chapters in the book.

A Brief Introduction to Current Cancer Gene Therapy.

Gene therapy has started in the late 1980s as novel, clinically applicable therapeutic option. It revolutionized the treatment of genetic diseases with the initial intent to repair or replace defective genes. Gene therapy has been adapted for treatment of malignant diseases to improve the outcome of cancer patients. In fact, cancer gene therapy has rapidly gained great interest and evolved into a research field with highest proportion of research activities in gene therapy. In this context, cancer gene therapy has long entered translation into clinical trials and therefore more than two-thirds of all gene therapy trials worldwide are aiming at the treatment of cancer disease using different therapeutic strategies. During the decades in cancer gene therapy, tremendous knowledge has accumulated. This led to significant improvements in vector design, transgene repertoire, more targeted interventions, use of novel gene therapeutic technologies such as CRISPR/Cas, sleeping beauty vectors, and development of effective cancer immunogene therapies. In this chapter, a brief overview of current key developments in cancer gene therapy is provided to gain insights into the recent directions in research as well as in clinical application of cancer gene therapy.

Cell-derived extracellular vesicles for CRISPR/Cas9 delivery: engineering strategies for cargo packaging and loading.

Genome editing technology has emerged as a potential therapeutic tool for treating incurable diseases. In particular, the discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems and the design of single-guide RNAs (sgRNAs) have revolutionized genome editing applications. Unfortunately, compared with the rapid development of gene-editing tools, the progress in the development of delivery technologies is lagging behind and thus limiting the clinical application of genome editing. To overcome these limitations, researchers have investigated various delivery systems, including viral and non-viral vectors for delivering CRISPR/Cas and sgRNA complexes. As natural endogenous nanocarriers, extracellular vesicles (EVs) present advantages of biocompatibility, low immunogenicity, stability, and high permeability, making them one of the most promising drug delivery vehicles. This review provides an overview of the fundamental mechanisms of EVs from the aspects of biogenesis, trafficking, cargo delivery, and function as nanotherapeutic agents. We also summarize the latest trends in EV-based CRISPR/Cas delivery systems and discuss the prospects for future development. In particular, we put our emphasis on the state-of-the-art engineering strategies to realize efficient cargo packaging and loading. Altogether, EVs hold promise in bridging genome editing in the laboratory and clinical applications of gene therapies by providing a safe, effective, and targeted delivery vehicle.

Application of gene therapy for reversing T-cell dysfunction in cancer

T cells are the key players of antitumor function that sense antigens produced by tumor cells and specifically target cancer cells to induce cell death. Therefore, it is widely understood that T cells are critical for cancer immunotherapy. Many recent studies discovered that T cells enter a dysfunctional or inactivated state. Cancer cells induce functional impairment of T cells by sustained expression of inhibitory receptors in T cells. Therefore, reversing T-cell dysfunction is critical for increasing the efficacy of tumor immunotherapy. In this research, we analyzed the cellular inhibition and activation of T cells using two cell line models: Jurkat (T cells) and MDA-MB-231 (breast cancer cells). We hypothesized that the knockdown of T-cell inhibition-causing genes might reverse the T-cell dysfunction caused by cancer cells. Therefore, we targeted Proprotein Convertase Subtilisin/Kexin Type 1 (PCSK1) gene in T cells and programmed death-ligand 1 (CD274) in cancer cells. We used small interference RNA (siRNA) to knock down the target gene. We used four μg/ml of lipopolysaccharide (LPS) to activate Jurkat cells. MDA-MB-231 co-culture with Jurkat cells reduced IL-2 expression level on Jurkat cells. We found that siRNA targeting CD274 in MDA-MB-231 reversed the reduced IL-2 expression level on Jurkat induced by Jurkat and MDA-MB-231 co-culture. Also, when the PCSK1 knockdown Jurkat cells and CD274 knockdown MDA-MB-231 were co-cultured, the most effective recovery of IL-2 expression level in Jurkat cells was observed. Overall, this study provides a novel strategy for reactivating T cells whose activation has been suppressed from cancer cells.

Polymer-based non-viral vectors for gene therapy in the skin

Gene therapy has emerged as a technique with the potential to treat human diseases; however, examples of the topical application of gene therapy as treatment for skin diseases remain scarce due to difficulties associated with the nature of the skin.

Delivery of DNA into Human Cells by Functionalized Lignin Nanoparticles.

Lignin is an aromatic plant cell wall polymer that is generated in large quantities as a low-value by-product by the pulp and paper industry and by biorefineries that produce renewable fuels and chemicals from plant biomass. Lignin structure varies among plant species and as a function of the method used for its extraction from plant biomass. We first explored the impact of this variation on the physico-chemical properties of lignin nanoparticles (LNPs) produced via a solvent exchange procedure and then examined whether LNPs produced from industrial sources of lignin could be used as delivery vehicles for DNA. Spherical LNPs were formed from birch and wheat BioLignin™ and from poplar thioglycolic acid lignin after dissolving the lignin in tetrahydrofuran (THF) and dialyzing it against water. Dynamic light scattering indicated that the diameter of these LNPs was dependent on the initial concentration of the lignin, while electrophoretic light scattering indicated that the LNPs had a negative zeta potential, which became less negative as the diameter increased. The dynamics of LNP formation as a function of the initial lignin concentration varied as a function of the source of the lignin, as did the absolute value of the zeta potential. After coating the LNPs with cationic poly-l-lysine, an electrophoretic mobility shift assay indicated that DNA could adsorb to LNPs. Upon transfection of human A549 lung carcinoma basal epithelial cells with functionalized LNPs carrying plasmid DNA encoding the enhanced green fluorescent protein (eGFP), green foci were observed under the microscope, and the presence of eGFP in the transfected cells was confirmed by ELISA. The low cytotoxicity of these LNPs and the ability to tailor diameter and zeta potential make these LNPs of interest for future gene therapy applications.

1014 – CELLULAR SENESCENCE: AN UNINTENDED TWIST FOR GENE AND CELL THERAPY APPLICATIONS

The success of Hematopoietic Stem and Progenitor Cell (HSPC) gene therapies critically depends on the capacity to genetically engineer HSPCs without compromising their functional properties. Emerging evidence, including our own, indicates that HSPC exposure to currently available gene transfer and gene editing technologies –which require prolonged ex-vivo culture, high viral vector doses and nuclease-induced DNA double strand breaks¬– unexpectedly converge on the DNA damage response (DDR), a signaling cascade leading to cell cycle arrest. Protracted DDR impairs the hematopoietic reconstitution of gene-modified cells upon transplantation; instead, its transient inhibition significantly improves their functionality. This experimental evidence uncovers a previously unappreciated interplay between viral vector sensing and the host cell DDR machinery in human HSPCs. Yet, we have just scratched the surface of a plethora of still unexplored cellular programs potentially hampering the clonal composition and the dynamics of reconstitution upon transplantation of this primitive cell compartment. We build on the innovative concept that current gene engineering protocols may inadvertently trigger the activation of a cellular senescence program in HSPCs with both cell-autonomous and paracrine short- and long-term consequences on engineered human hematopoiesis. By employing quantitative imaging and cutting-edge genomic technologies and taking advantage of uniquely available gene therapy platforms and primary patient samples, we aim to identify the molecular determinants that promote senescence in HSPCs and design innovative hypothesis-driven strategies to mitigate senescence barriers for more effective, safer and clinically broader gene therapy applications. Our findings will unveil a new set of mechanisms controlling the biology of gene-engineered HSPCs and open novel scientific horizons for the development of innovative gene and cell therapies. The success of Hematopoietic Stem and Progenitor Cell (HSPC) gene therapies critically depends on the capacity to genetically engineer HSPCs without compromising their functional properties. Emerging evidence, including our own, indicates that HSPC exposure to currently available gene transfer and gene editing technologies –which require prolonged ex-vivo culture, high viral vector doses and nuclease-induced DNA double strand breaks¬– unexpectedly converge on the DNA damage response (DDR), a signaling cascade leading to cell cycle arrest. Protracted DDR impairs the hematopoietic reconstitution of gene-modified cells upon transplantation; instead, its transient inhibition significantly improves their functionality. This experimental evidence uncovers a previously unappreciated interplay between viral vector sensing and the host cell DDR machinery in human HSPCs. Yet, we have just scratched the surface of a plethora of still unexplored cellular programs potentially hampering the clonal composition and the dynamics of reconstitution upon transplantation of this primitive cell compartment. We build on the innovative concept that current gene engineering protocols may inadvertently trigger the activation of a cellular senescence program in HSPCs with both cell-autonomous and paracrine short- and long-term consequences on engineered human hematopoiesis. By employing quantitative imaging and cutting-edge genomic technologies and taking advantage of uniquely available gene therapy platforms and primary patient samples, we aim to identify the molecular determinants that promote senescence in HSPCs and design innovative hypothesis-driven strategies to mitigate senescence barriers for more effective, safer and clinically broader gene therapy applications. Our findings will unveil a new set of mechanisms controlling the biology of gene-engineered HSPCs and open novel scientific horizons for the development of innovative gene and cell therapies.

Cancer Gene Therapy: Development and Production of Lentiviral Vectors for Gene Therapy.

Lentiviral vectors are among the most used vectors in gene therapy to treat pathologies of different origins, such as cancers, rare monogenic diseases or neurological disorders. This chapter provides an overview on lentiviral vector developments in terms of vector design and manufacture for gene therapy applications. The state of the art of vector production will be summarized face to the recent developments contributing to improve vector safety, efficacy and manufacturing robustness, focusing on human immunodeficiency virus 1 (HIV-1) based lentiviral vectors. Transient and stable production systems will be discussed highlighting recent advances in producer cell line development. Challenges in lentiviral vector development upstream and downstream will be addressed with a particular focus on the improvements undertaken to increase vector yields and production scalability.

Polyethyleneimine-Based Lipopolyplexes as Carriers in Anticancer Gene Therapies.

Recent years have witnessed rapidly growing interest in application of gene therapies for cancer treatment. However, this strategy requires nucleic acid carriers that are both effective and safe. In this context, non-viral vectors have advantages over their viral counterparts. In particular, lipopolyplexes—nanocomplexes consisting of nucleic acids condensed with polyvalent molecules and enclosed in lipid vesicles—currently offer great promise. In this article, we briefly review the major aspects of developing such non-viral vectors based on polyethyleneimine and outline their properties in light of anticancer therapeutic strategies. Finally, examples of current in vivo studies involving such lipopolyplexes and possibilities for their future development are presented.

Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina.

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of AAV transduction is usually critical, which affects the overall experimental outcomes. One of the main determinants for transduction efficiency is the serotype or variant of the AAV vector. Currently, various artificial AAV serotypes and variants are available with different affinities to host cell surface receptors. For retinal gene therapy, this results in varying degrees of transduction efficiencies for different retinal cell types. In addition, the injection route and the quality of AAV production may also affect the retinal AAV transduction efficiencies. Therefore, it is essential to compare the efficiency of different variants, batches, and methodologies. The digital droplet PCR (dd-PCR) method quantifies the nucleic acids with high precision and allows performing absolute quantification of a given target without any standard or a reference. Using dd-PCR, it is also feasible to assess the transduction efficiencies of AAVs by absolute quantification of AAV genome copy numbers within an injected retina. Here, we provide a straightforward method to quantify the transduction rate of AAVs in retinal cells using dd-PCR. With minor modifications, this methodology can also be the basis for the copy number quantification of mitochondrial DNA as well as assessing the efficiency of base editing, critical for several retinal diseases and gene therapy applications.

Comparison of highly pure rAAV9 vector stocks produced in suspension by PEI transfection or HSV infection reveals striking quantitative and qualitative differences

Recent clinical successes have propelled recombinant adeno-associated virus vectors (rAAV) to the center stage for human gene therapy applications. However, the exploding demand for high titers of highly pure rAAV vectors for clinical applications and market needs remains hindered by challenges met at the manufacturing stage. The production of rAAV by transfection in suspension cells remains one of the most commonly used production platforms. In this study, we describe our optimized protocol to produce rAAV by polyethyleneimine (PEI)-mediated transfection in suspension HEK293 cells, along with a side-by-side comparison to our high-performing system using the herpes simplex virus (HSV). Further, we detail a new, robust, and highly efficient downstream purification protocol compatible with both transfection and infection-based harvests that generated rAAV9 stocks of high purity. Our in-depth comparison revealed quantitative, qualitative, and biological differences between PEI-mediated transfection and HSV infection. The HSV production system yielded to higher rAAV vector titers, higher specific yields, and a higher percentage of full capsids than transfection. Furthermore, HSV-produced stocks had a significantly lower concentration of residual host cell proteins and helper DNA impurities, but contained detectable levels of HSV DNA. Importantly, the potency of PEI-produced and HSV-produced rAAV stocks were identical. Analyses of AAV Rep and Cap expression levels and replication showed that HSV-mediated production led to a lower expression of Rep and Cap, but increased levels of AAV genome replication. Our methodology enables high-yield, high purity rAAV production and a biological framework to improve transfection quality and yields by mimicking HSV-induced biological outcomes.

Synthetic biology approach to developing all-in-one baculovirus vector using mammalian introns and miRNA binding sites

BackgroundInsect baculovirus is a promising vector for gene delivery into mammalian cells. We developed a Cre/loxP-based hybrid baculovirus comprising two viruses: one expressing Cre recombinase and the other harboring the transgene flanked by two loxP sites. Co-transduction of mammalian cells with two viruses confers Cre expression, which excises the loxP-flanking cassette off baculovirus genome and catalyzes DNA minicircle formation, thereby prolonging transgene expression. Two separate baculoviruses avoid undesirable recombination and loss of transgene during virus production process, but reduces gene delivery efficiency and complicates applications. MethodsTo tackle this problem, we exploited synthetic biology strategy to control Cre expression and develop an all-in-one baculovirus harboring both cre and loxP-flanking transgene cassette. We controlled cre transcription with a mammalian EF-1α promoter, and regulated cre translation with an intron inserted within the coding region and bantam miRNA binding site at the 3′UTR. Significant findingsThe all-in-one baculovirus vector selectively conferred Cre/loxP-mediated recombination in mammalian cells, but not in E. coli and Sf-9 cells, hence circumventing transgene excision from the virus genome during gene cloning in E. coli and virus amplification in insect cells. The all-in-on vector enabled formation of DNA minicircle in mammalian cells at efficiencies exceeding 80% and implicated its potential for gene delivery. Our design may be expanded to other gene delivery systems that require two separate vectors and is beneficial to gene therapy applications.

Efficient Generation of P53 Biallelic Mutations in Diannan Miniature Pigs Using RNA-Guided Base Editing.

The base editing 3 (BE3) system, a single-base gene editing technology developed using CRISPR/Cas9n, has a broad range of applications for human disease model construction and gene therapy, as it is highly efficient, accurate, and non-destructive. P53 mutations are present in more than 50% of human malignancies. Due to the similarities between humans and pigs at the molecular level, pig models carrying P53 mutations can be used to research the mechanism of tumorigenesis and improve tumor diagnosis and treatment. According to pathogenic mutations of the human P53 gene at W146* and Q100*, sgRNAs were designed to target exon 4 and exon 5 of the porcine P53 gene. The target editing efficiencies of the two sgRNAs were 61.9% and 50.0%, respectively. The editing efficiency of the BE3 system was highest (about 60%) when C (or G) was at the 5th base. Puromycin screening revealed that 75.0% (21/28) and 68.7% (22/32) of cell colonies contained a P53 mutation at sgRNA-Exon5 and sgRNA-Exon4, respectively. The reconstructed embryos from sgRNA-Exon5-5# were transferred into six recipient gilts, all of which aborted. The reconstructed embryos from sgRNA-Exon4-7# were transferred into 6 recipient gilts, 3 of which became pregnant, resulting in 14 live and 3 dead piglets. Sequencing analyses of the target site confirmed 1 P53 monoallelic mutation and 16 biallelic mutations. The qPCR analysis showed that the P53 mRNA expression level was significantly decreased in different tissues of the P53 mutant piglets (p < 0.05). Additionally, confocal microscopy and western blot analysis revealed an absence of P53 expression in the P53 mutant fibroblasts, livers, and lung tissues. In conclusion, a porcine cancer model with a P53 point mutation can be obtained via the BE3 system and somatic cell nuclear transfer (SCNT).

Leveraging high-throughput purification to accelerate viral vector process development

Recombinant adeno-associated virus (AAV) has been broadly used as a delivery tool for gene therapy applications. The development of a robust purification process is essential for delivering high purity and quality AAV products to clinic. The short clinical timelines and material limitations of early-stage development pose unique challenges to developing robust and scalable downstream purification processes. One approach to overcome these limitations is to leverage high throughput (HTP) strategies and automation technologies for purification process development, an approach that is well established in protein biologics and other areas. However, due to the unique challenges related to viral vector purification, implementing HTP approaches for gene therapy process development has not been explored extensively. In this paper, we established a HTP chromatography platform and demonstrated its capability to facilitate gene therapy purification process development using both mini-columns and self-packed resin plates. The end-to-end development workflow for AAV HTP purification is detailed in this work with the expectation of serving as an introductory for the AAV purification development field. Comparable process performance was confirmed between a bench-scale chromatography process and an HTP chromatography format. Slightly lower recovery was observed using the HTP format (62% vs 75%), as well as %full capsid enrichment (71% vs. 82%). Comparable impurity clearance capability was demonstrated between the two different systems as well. It was concluded that the established HTP chromatography formats can serve as a surrogate to bench-scale chromatography development to reduce material needs and development timelines for AAV purification development.

Curative therapy for hemoglobinopathies: an International Society for Cell & Gene Therapy Stem Cell Engineering Committee review comparing outcomes, accessibility and cost of ex vivo stem cell gene therapy versus allogeneic hematopoietic stem cell transplantation

Thalassemia and sickle cell disease (SCD) are the most common monogenic diseases in the world and represent a growing global health burden. Management is limited by a paucity of disease-modifying therapies; however, allogeneic hematopoietic stem cell transplantation (HSCT) and autologous HSCT after genetic modification offer patients a curative option. Allogeneic HSCT is limited by donor selection, morbidity and mortality from transplant conditioning, graft-versus-host disease and graft rejection, whereas significant concerns regarding long-term safety, efficacy and cost limit the broad applicability of gene therapy. Here the authors review current outcomes in allogeneic and autologous HSCT for transfusion-dependent thalassemia and SCD and provide our perspective on issues surrounding accessibility and costs as barriers to offering curative therapy to patients with hereditary hemoglobinopathies.

4D-Nucleofector®️ LV Unit: Efficient, non-viral large-scale transfection for cell and gene therapy applications

<h3>Watch the video or read the poster to learn:</h3> <ul class="focus-visible" data-focus-visible-added=""><li data-stringify-indent="0" data-stringify-border="0">An electroporation platform for efficient, non-viral large-scale transfection for cell and gene therapy applications</li><li data-stringify-indent="0" data-stringify-border="0">How Nucleofector(R) technology can be applied at scales from 10,000 to 1 billion cells</li><li> </li><li data-stringify-indent="0" data-stringify-border="0">Five key considerations for success in genetic modification for cell therapy</li></ul><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Timo-Gleissner_Lonza copy.jpg" data-image="1"></div><div class="author-det"> <b>Timo Gleissner</b> is Associate Director Engineering within Bioscience Solutions at Lonza Cologne GmbH and responsible for the technical development of Lonza’s Nucleofector^®️ Transfection Platforms. In his current role Timo lead the development of the 4D-Nucleofector^®️ LV Unit for large-scale transfection and had the technical responsibility for the product design of the 21CFR part 11 compliant software as well as the TheraPEAK^®️ Consumables for GMP manufacturing. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Andrea-Toell copy.jpg" data-image="1"></div><div class="author-det"> <b>Andrea Toell</b> is a Senior Product Manager at Lonza Cologne GmbH and is responsible for Lonza’s Nucleofector^®️ Transfection Portfolio. In her current role she is focusing on large volume transfection and cell and gene therapy applications (CAR-T cells, genome editing, GMP manufacturing). In her 20 years’ experience with the technology she gained a broad knowledge about transfection technologies and factors that are important for successful transfection experiments. </div></div></div>

In vitro functional genetic modification of canine adenovirus type 2 genome by CRISPR/Cas9

AbstractGenetically modified oncolytic adenoviruses have been proposed as a vehicle for cancer therapy. However, several concerns, such as toxicity to normal cells and organs, lack of suitable cell surface receptors to allow viral entry to the desired cell type(s), and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of adenoviral-mediated cancer gene therapy. Successful virotherapy will require efficient transductional and transcriptional targeting to enhance therapeutic efficacy by ensuring targeted adenoviral infection, replication, and/or therapeutic transgene expression. Targeted modification of viral components, such as viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is complex, time consuming, and expensive. It is dependent on the presence of unique restriction enzyme sites that may or may not be present in the target location. Clustered regularly interspaced short palindromic repeat (CRISPR) along with the RNA-guided nuclease Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in a variety of cells and organisms. However, the ability of the CRISPR/Cas9 system to precisely and efficiently make genetic modification, as well as introduce gene replacements, in adenoviral genomes, remains essentially unknown. Herein the ability of in vitro CRISPR/CAS9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification of the viral genome was assessed. To demonstrate the feasibility of this goal, CRISPR/Cas9 has been used to successfully insert the RFP (red fluorescent protein) reporter construct into the CAV2 genome. Initial results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. Furthermore, this application was expanded, using multiple guide RNAs, to conduct gene replacement in the CAV2 genome by substituting a portion of the E3 gene with a construct designed to express a single chain antibody to canine PD-1. Thus, this work provides a significantly improved and efficient method for targeted editing of adenoviruses to generate altered and potentially therapeutic viral genomes in the shortest possible time.

Hutchinson-Gilford Progeria Syndrome (Hgps) and Application of Gene Therapy Based Crispr/Cas Technology as A Promising Innovative Treatment Approach

Hutchinson-Gilford progeria syndrome (HGPS), also known as progeria of childhood or progeria is a rare, rapid, autosomal dominant genetic disorder characterized by premature aging which occurs shortly after birth. HGPS occurs as a result of de novo point mutation in the gene recognized as LMNA gene that encodes two proteins, Lamin A protein and Lamin C protein which are the structural components of the nuclear envelope. Mutations in the gene trigger abnormal splicing and induce internal deletion of 50 amino acids leading to the development of a truncated form of Lamin A protein known as Progerin. Progerin generation can be considered the crucial step in HGPS since the protein is highly toxic to human cells, permanently farnesylated, and exhibits variation in several biochemical and structural properties within the individual. HGPS also produces complications such as skin alterations, growth failure, atherosclerosis, hair and fat loss, and bone and joint diseases. We have also revised all relevant patents relating to Hutchinson-Gilford progeria syndrome and its therapy in the current article. The goal of the present review article is to provide information about Hutchinson- Gilford progeria syndrome (HGPS) and the use of CRISPR/Cas technology as a promising treatment approach in the treatment of the disease. The review also discusses about different pharmacological and non-pharmacological methods of treatment currently used for HGPS. The main limitation associated with progeria is the lack of a definitive cure. The existing treatment modality provides only symptomatic relief. Therefore, it is high time to develop a therapeutic method that hastens premature aging in such patients. CRISPR/Cas technology is a novel gene-editing tool that allows genome editing at specific loci and is found to be a promising therapeutic approach for the treatment of genetic disorders such as HGPS where dominant-negative mutations take place.

Improved lentiviral vector titers from a multi-gene knockout packaging line

Lentiviral vectors (LVs) are robust delivery vehicles for gene therapy as they can efficiently integrate transgenes into host cell genomes. However, LVs with lengthy or complex expression cassettes typically are produced at low titers and have reduced gene transfer capacity, creating barriers for clinical and commercial applications. Modifications of the packaging cell line and methods may be able to produce complex vectors at higher titer and infectivity and may improve production of many different LVs. In this study, we identified two host restriction factors in HEK293T packaging cells that impeded LV production, 2'-5'-oligoadenylate synthetase 1 (OAS1) and low-density lipoprotein receptor (LDLR). Knocking out these two genes separately led to ∼2-fold increases in viral titer. We created a monoclonal cell line, CRISPRed HEK293T to Disrupt Antiviral Response (CHEDAR), by successively knocking out OAS1, LDLR, and PKR, a previously identified factor impeding LV titers. Packaging in CHEDAR yielded ∼7-fold increases in physical particles, full-length vector RNA, and vector titers. In addition, overexpressing transcription elongation factors, SPT4 and SPT5, during packaging improved the production of full-length vector RNA, thereby increasing titers by ∼2-fold. Packaging in CHEDAR with over-expression of SPT4 and SPT5 led to ∼11-fold increases of titers. These approaches improved the production of a variety of LVs, especially vectors with low titers or with internal promoters in the reverse orientation, and may be beneficial for multiple gene therapy applications.