RESUMO
Natural killer (NK) cells have high intrinsic cytotoxic capacity, and clinical trials have demonstrated their safety and efficacy for adoptive cancer therapy. Expression of chimeric antigen receptors (CARs) enhances NK cell target specificity, with these cells applicable as off-the-shelf products generated from allogeneic donors. Here, we present for the first time an innovative approach for CAR NK cell engineering employing a non-viral Sleeping Beauty (SB) transposon/transposase-based system and minimized DNA vectors termed minicircles. SB-modified peripheral blood-derived primary NK cells displayed high and stable CAR expression and more frequent vector integration into genomic safe harbors than lentiviral vectors. Importantly, SB-generated CAR NK cells demonstrated enhanced cytotoxicity compared with non-transfected NK cells. A strong antileukemic potential was confirmed using established acute lymphocytic leukemia cells and patient-derived primary acute B cell leukemia and lymphoma samples as targets in vitro and in vivo in a xenograft leukemia mouse model. Our data suggest that the SB-transposon system is an efficient, safe, and cost-effective approach to non-viral engineering of highly functional CAR NK cells, which may be suitable for cancer immunotherapy of leukemia as well as many other malignancies.
Assuntos
Vetores Genéticos , Imunoterapia Adotiva , Células Matadoras Naturais , Receptores de Antígenos Quiméricos , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Animais , Camundongos , Vetores Genéticos/genética , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Imunoterapia Adotiva/métodos , Ensaios Antitumorais Modelo de Xenoenxerto , Transposases/genética , Transposases/metabolismo , Linhagem Celular Tumoral , Elementos de DNA Transponíveis , Citotoxicidade Imunológica , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Engenharia Celular/métodosRESUMO
The ability to target the native production site of factor VIII (FVIII)-liver sinusoidal endothelial cells (LSECs)-can improve the outcome of hemophilia A (HA) gene therapy. By testing a matrix of ultrasound-mediated gene delivery (UMGD) parameters for delivering a GFP plasmid into the livers of HA mice, we were able to define specific conditions for targeted gene delivery to different cell types in the liver. Subsequently, two conditions were selected for experiments to treat HA mice via UMGD of an endothelial-specific human FVIII plasmid: low energy (LE; 50 W/cm2, 150 µs pulse duration) to predominantly target endothelial cells or high energy (HE; 110 W/cm2, 150 µs pulse duration) to predominantly target hepatocytes. Both groups of UMGD-treated mice achieved persistent FVIII activity levels of â¼10% over 84 days post treatment; however, half of the HE-treated mice developed low-titer inhibitors while none of the LE mice did. Plasma transaminase levels and histological liver examinations revealed minimal transient liver damage that was lower in the LE group than in the HE group. These results indicate that UMGD can safely target LSECs with a lower-energy condition to achieve persistent FVIII gene expression, demonstrating that this novel technology is highly promising for therapeutic correction of HA.
Assuntos
Fator VIII , Hemofilia A , Humanos , Fator VIII/metabolismo , Hemofilia A/genética , Hemofilia A/terapia , Hemofilia A/patologia , Células Endoteliais/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Terapia Genética/métodosRESUMO
Heart disease is the leading cause of human deaths worldwide. Due to lacking cardiomyocytes with replicative capacity and cardiac progenitor cells with differentiation potential in adult hearts, massive loss of cardiomyocytes after ischemic events produces permanent damage, ultimately leading to heart failure. Cellular reprogramming is a promising strategy to regenerate heart by induction of cardiomyocytes from other cell types, such as cardiac fibroblasts. In contrast to conventional virus-based cardiac reprogramming, non-viral approaches greatly reduce the potential risk that includes disruption of genome integrity by integration of foreign DNAs, expression of exogenous genes with oncogenic potential, and appearance of partially reprogrammed cells harmful for the physiological functions of tissues/organs, which impedes their in-vivo applications. Here, we review the recent progress in development of non-viral approaches to directly reprogram somatic cells towards cardiomyocytes and their therapeutic application for heart regeneration.
Assuntos
Reprogramação Celular/fisiologia , Miócitos Cardíacos/metabolismo , Medicina Regenerativa/métodos , Animais , Humanos , CamundongosRESUMO
With the emergence of gene therapy utilizing viral vectors, the potential risks associated with these vectors have prompted increased attention toward non-viral alternatives. DNA nanotechnology enables the assembly of specific oligonucleotide chains into nanostructures possessing defined spatial configurations. Due to their inherent characteristics, DNA nanostructures possess natural advantages as carriers for regulating gene expression in a non-viral manner. Cholesterol modification can convert DNA nanostructures from hydrophilic materials to amphiphilic materials, thereby extending their systemic circulation time. In this study, the high-dimensional design and cholesterol modification are shown to prolong the systemic circulation half-life of DNA nanostructures in mice. Specifically, the tetrahedron structure modified with three cholesterol molecules (TDN-3Chol) exhibit excellent circulation time and demonstrate a preference for renal uptake. The unique characteristics of TDN-3Chol can effectively deliver p53 siRNA to the mouse renal tubular tissue, resulting in successful knockdown of p53 and demonstrating its potential for preventing acute kidney injury. Furthermore, TDN-3Chol is not exhibited significant toxicity in mice, highlighting its promising role as a non-viral vector for targeted gene expression regulation in the kidneys. The designed non-viral vector as a prophylactic medication shows potential in addressing the current clinical challenges associated with nephrotoxic drugs.
Assuntos
Injúria Renal Aguda , Colesterol , DNA , Rim , Nanoestruturas , RNA Interferente Pequeno , Animais , Nanoestruturas/química , Injúria Renal Aguda/prevenção & controle , Colesterol/química , DNA/química , Rim/metabolismo , Camundongos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Técnicas de Transferência de GenesRESUMO
Gene therapy uses modern molecular biology methods to repair disease-causing genes. As a burgeoning therapeutic, it has been widely applied for cancer therapy. Since 1989, there have been numerous clinical gene therapy cases worldwide. However, a few are successful. The main challenge of clinical gene therapy is the lack of efficient and safe vectors. Although viral vectors show high transfection efficiency, their application is still limited by immune rejection and packaging capacity. Therefore, the development of non-viral vectors is overwhelming. Nanoplatform-based non-viral vectors become a hotspot in gene therapy. The reasons are mainly as follows. 1) Non-viral vectors can be engineered to be uptaken by specific types of cells or tissues, providing effective targeting capability. 2) Non-viral vectors can protect goods that need to be delivered from degradation. 3) Nanoparticles can transport large-sized cargo such as CRISPR/Cas9 plasmids and nucleoprotein complexes. 4) Nanoparticles are highly biosafe, and they are not mutagenic in themselves compared to viral vectors. 5) Nanoparticles are easy to scale preparation, which is conducive to clinical conversion and application. Here, an overview of the categories of nanoplatform-based non-viral gene vectors, the limitations on their development, and their applications in cancer therapy.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética , Nanopartículas , Neoplasias , Humanos , Neoplasias/terapia , Neoplasias/genética , Terapia Genética/métodos , Nanopartículas/química , Animais , Vetores Genéticos/genéticaRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) have garnered significant interest for their tumor-tropic property, making them potential therapeutic delivery vehicles for cancer treatment. We have previously shown the significant anti-tumour activity in mice preclinical models and companion animals with naturally occurring cancers using non-virally engineered MSCs with a therapeutic transgene encoding cytosine deaminase and uracil phosphoribosyl transferase (CDUPRT) and green fluorescent protein (GFP). Clinical studies have shown improved response rate with combinatorial treatment of 5-fluorouracil and Interferon-beta (IFNb) in peritoneal carcinomatosis (PC). However, high systemic toxicities have limited the clinical use of such a regime. METHODS: In this study, we evaluated the feasibility of intraperitoneal administration of non-virally engineered MSCs to co-deliver CDUPRT/5-Flucytosine prodrug system and IFNb to potentially enhance the cGAS-STING signalling axis. Here, MSCs were engineered to express CDUPRT or CDUPRT-IFNb. Expression of CDUPRT and IFNb was confirmed by flow cytometry and ELISA, respectively. The anti-cancer efficacy of the engineered MSCs was evaluated in both in vitro and in vivo model. ES2, HT-29 and Colo-205 were cocultured with engineered MSCs at various ratio. The cell viability with or without 5-flucytosine was measured with MTS assay. To further compare the anti-cancer efficacy of the engineered MSCs, peritoneal carcinomatosis mouse model was established by intraperitoneal injection of luciferase expressing ES2 stable cells. The tumour burden was measured through bioluminescence tracking. RESULTS: Firstly, there was no changes in phenotypes of MSCs despite high expression of the transgene encoding CDUPRT and IFNb (CDUPRT-IFNb). Transwell migration assays and in-vivo tracking suggested the co-expression of multiple transgenes did not impact migratory capability of the MSCs. The superiority of CDUPRT-IFNb over CDUPRT expressing MSCs was demonstrated in ES2, HT-29 and Colo-205 in-vitro. Similar observations were observed in an intraperitoneal ES2 ovarian cancer xenograft model. The growth of tumor mass was inhibited by ~ 90% and 46% in the mice treated with MSCs expressing CDUPRT-IFNb or CDUPRT, respectively. CONCLUSIONS: Taken together, these results established the effectiveness of MSCs co-expressing CDUPRT and IFNb in controlling and targeting PC growth. This study lay the foundation for the development of clinical trial using multigene-armed MSCs for PC.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Pentosiltransferases , Neoplasias Peritoneais , Transgenes , Animais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Neoplasias Peritoneais/terapia , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/patologia , Humanos , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Linhagem Celular Tumoral , Interferon beta/metabolismo , Interferon beta/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Camundongos , FemininoRESUMO
Over the past twenty years, ocular gene therapy has primarily focused on addressing diseases linked to various genetic factors. The eye is an ideal candidate for gene therapy due to its unique characteristics, such as easy accessibility and the ability to target both corneal and retinal conditions, including retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), and Stargardt disease. Currently, literature documents 33 clinical trials in this field, with the most promising results emerging from trials focused on LCA. These successes have catalyzed further research into other ocular conditions such as glaucoma, AMD, RP, and choroideremia. The effectiveness of gene therapy relies on the efficient delivery of genetic material to specific cells, ensuring sustained and optimal gene expression over time. Viral vectors have been widely used for this purpose, although concerns about potential risks such as immune reactions and genetic mutations have led to the development of non-viral vector systems. Preliminary laboratory research and clinical investigations have shown a connection between vector dosage and the intensity of immune response and inflammation in the eye. The method of administration significantly influences these reactions, with subretinal delivery resulting in a milder humoral response compared to the intravitreal route. This review discusses various ophthalmic diseases, including both corneal and retinal conditions, and their underlying mechanisms, highlighting recent advances and applications in ocular gene therapies.
Assuntos
Terapia Genética , Vetores Genéticos , Humanos , Terapia Genética/métodos , Oftalmopatias/terapia , Oftalmopatias/genética , Técnicas de Transferência de Genes , Doenças Retinianas/terapia , Doenças Retinianas/genética , AnimaisRESUMO
Cellular therapies have the potential to advance treatment for a broad array of diseases but rely on viruses for genetic reprogramming. The time and cost required to produce viruses has created a bottleneck that constricts development of and access to cellular therapies. Electroporation is a non-viral alternative for genetic reprogramming that bypasses these bottlenecks, but current electroporation technology suffers from low throughput, tedious optimization, and difficulty scaling to large-scale cell manufacturing. Here, we present an adaptable microfluidic electroporation platform with the capability for rapid, multiplexed optimization with 96-well plates. Once parameters are optimized using small volumes of cells, transfection can be seamlessly scaled to high-volume cell manufacturing without re-optimization. We demonstrate optimizing transfection of plasmid DNA to Jurkat cells, screening hundreds of different electrical waveforms of varying shapes at a speed of ~3 s per waveform using ~20 µL of cells per waveform. We selected an optimal set of transfection parameters using a low-volume flow cell. These parameters were then used in a separate high-volume flow cell where we obtained similar transfection performance by design. This demonstrates an alternative non-viral and economical transfection method for scaling to the volume required for producing a cell therapy without sacrificing performance. Importantly, this transfection method is disease-agnostic with broad applications beyond cell therapy.
Assuntos
Eletroporação , Microfluídica , Humanos , Transfecção , Terapia Baseada em Transplante de Células e Tecidos , EletricidadeRESUMO
Recent advancements have focused on enhancing factor VIII half-life and refining its delivery methods, despite the well-established knowledge that factor VIII deficiency is the main clotting protein lacking in hemophilia. Consequently, both viral and non-viral delivery systems play a crucial role in enhancing the quality of life for hemophilia patients. The utilization of viral vectors and the manipulation of non-viral vectors through targeted delivery are significant advancements in the field of cellular and molecular therapies for hemophilia. These developments contribute to the progression of treatment strategies and hold great promise for improving the overall well-being of individuals with hemophilia. This review study comprehensively explores the application of viral and non-viral vectors in cellular (specifically T cell) and molecular therapy approaches, such as RNA, monoclonal antibody (mAb), and CRISPR therapeutics, with the aim of addressing the challenges in hemophilia treatment. By examining these innovative strategies, the study aims to shed light on potential solutions to enhance the efficacy and outcomes of hemophilia therapy.
Assuntos
Hemofilia A , Humanos , Hemofilia A/terapia , Hemofilia A/genética , Qualidade de Vida , Terapia Genética/métodos , Fator VIII , Fatores de Coagulação Sanguínea , Vetores GenéticosRESUMO
The rise of gene therapy has solved many diseases that cannot be effectively treated by conventional methods. Gene vectors is very important to protect and deliver the therapeutic genes to the target site. Polyethyleneimine (PEI) modified with mannitol could enhance the gene transfection efficiency reported by our group previously. In order to further control and improve the effective gene release to action site, disulfide bonds were introduced into mannitol-modified PEI to construct new non-viral gene vectors PeiSM. The degrees of mannitol linking with disulfide bonds were screened. Among them, moderate mannitol-modified PEI with disulfide bonds showed the best transfection efficiency, and significantly enhanced long-term systemic transgene expression for 72 hin vivoeven at a single dose administration, and could promote caveolae-mediated uptake through up-regulating the phosphorylation of caveolin-1 and increase the loaded gene release from the nanocomplexes in high glutathione intracellular environment. This functionalized gene delivery system can be used as an potential and safe non-viral nanovector for further gene therapy.
Assuntos
Vetores Genéticos , Glutationa , Polietilenoimina , Transfecção , Polietilenoimina/química , Transfecção/métodos , Glutationa/metabolismo , Glutationa/química , Animais , Humanos , Vetores Genéticos/química , Vetores Genéticos/genética , Manitol/química , Camundongos , Caveolina 1/metabolismo , Caveolina 1/genética , Terapia Genética/métodos , Técnicas de Transferência de Genes , Dissulfetos/químicaRESUMO
Short interfering RNA (siRNA) therapeutics have soared in popularity due to their highly selective and potent targeting of faulty genes, providing a non-palliative approach to address diseases. Despite their potential, effective transfection of siRNA into cells requires the assistance of an accompanying vector. Vectors constructed from non-viral materials, while offering safer and non-cytotoxic profiles, often grapple with lackluster loading and delivery efficiencies, necessitating substantial milligram quantities of expensive siRNA to confer the desired downstream effects. We detail the recombinant synthesis of a diverse series of coiled-coil supercharged protein (CSP) biomaterials systematically designed to investigate the impact of two arginine point mutations (Q39R and N61R) and decahistidine tags on liposomal siRNA delivery. The most efficacious variant, N8, exhibits a twofold increase in its affinity to siRNA and achieves a twofold enhancement in transfection activity with minimal cytotoxicity in vitro. Subsequent analysis unveils the destabilizing effect of the Q39R and N61R supercharging mutations and the incorporation of C-terminal decahistidine tags on α-helical secondary structure. Cross-correlational regression analyses reveal that the amount of helical character in these mutants is key in N8's enhanced siRNA complexation and downstream delivery efficiency.
Assuntos
Histidina , Lipossomos , Oligopeptídeos , RNA Interferente Pequeno , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/administração & dosagem , Histidina/química , Histidina/genética , Humanos , Lipossomos/química , Oligopeptídeos/química , Oligopeptídeos/genética , Transfecção/métodos , Estrutura Secundária de ProteínaRESUMO
USH2A mutations are a common cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome, for which there are currently no approved treatments. Gene augmentation is a valuable therapeutic strategy for treating many inherited retinal diseases; however, conventional adeno-associated virus (AAV) gene therapy cannot accommodate cDNAs exceeding 4.7 kb, such as the 15.6-kb-long USH2A coding sequence. In the present study, we adopted an alternative strategy to successfully generate scaffold/matrix attachment region (S/MAR) DNA plasmid vectors containing the full-length human USH2A coding sequence, a GFP reporter gene, and a ubiquitous promoter (CMV or CAG), reaching a size of approximately 23 kb. We assessed the vectors in transfected HEK293 cells and USH2A patient-derived dermal fibroblasts in addition to ush2au507 zebrafish microinjected with the vector at the one-cell stage. pS/MAR-USH2A vectors drove persistent transgene expression in patient fibroblasts with restoration of usherin. Twelve months of GFP expression was detected in the photoreceptor cells, with rescue of Usher 2 complex localization in the photoreceptors of ush2au507 zebrafish retinas injected with pS/MAR-USH2A. To our knowledge, this is the first reported vector that can be used to express full-length usherin with functional rescue. S/MAR DNA vectors have shown promise as a novel non-viral retinal gene therapy, warranting further translational development.
Assuntos
Síndromes de Usher , Animais , Humanos , Síndromes de Usher/genética , Síndromes de Usher/terapia , Peixe-Zebra/genética , Células HEK293 , Mutação , DNA , Plasmídeos/genética , Proteínas da Matriz Extracelular/genéticaRESUMO
Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Camundongos , Ribonucleoproteínas/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Encéfalo/metabolismoRESUMO
Currently, many types of non-linear topological structure polymers, such as brush-shaped, star, branched and dendritic structures, have captured much attention in the field of gene delivery and nanomedicine. Compared with linear polymers, non-linear topological structural polymers offer many advantages, including multiple terminal groups, broad and complicated spatial architecture and multi-functionality sites to enhance gene delivery efficiency and targeting capabilities. Nevertheless, the complexity of their synthesis process severely hampers the development and applications of nonlinear topological polymers. This review aims to highlight various synthetic approaches of non-linear topological architecture polymers, including reversible-deactivation radical polymerization (RDRP) including atom-transfer radical polymerization (ATRP), nitroxide-mediated polymerization (NMP), reversible addition-fragmentation chain transfer (RAFT) polymerization, click chemistry reactions and Michael addition, and thoroughly discuss their advantages and disadvantages, as well as analyze their further application potential. Finally, we comprehensively discuss and summarize different non-linear topological structure polymers for genetic materials delivering performance both in vitro and in vivo, which indicated that topological effects and nonlinear topologies play a crucial role in enhancing the transfection performance of polymeric vectors. This review offered a promising guideline for the design and development of novel nonlinear polymers and facilitated the development of a new generation of polymer-based gene vectors.
Assuntos
Técnicas de Transferência de Genes , Polímeros , Polímeros/química , Transfecção , Química Click , PolimerizaçãoRESUMO
BACKGROUND: Research on prostate cancer is mostly performed using cell lines derived from metastatic disease, not reflecting stages of tumor initiation or early progression. Establishment of cancer cell lines derived from the primary tumor site has not been described so far. By definition, cancer cells are able to be cultured indefinitely, whereas normal epithelial cells undergo senescence in vitro. Epithelial cells can be immortalized, accomplished by using viral integration of immortalization factors. Viral approaches, however, might be impaired by regulatory and safety issues as well as random integration into regulatory genetic elements, modifying precise gene expression. We intend to use surgical specimen of prostate cancer patients to (i) prove for establishment of cancer cell lines, and (ii) perform non-viral, Sleeping Beauty (SB) transposase-based immortalization of prostate epithelial cells. METHODS: Radical prostatectomy samples of prostate cancer patients (n = 4) were dissociated and cultured in vitro. Cells were cultivated either without or after non-viral, Sleeping-Beauty transposase-based stable transfection with immortalization factors SV40LT and hTERT. Established cell lines were analyzed in vitro and in vivo for characteristics of prostate (cancer) cells. RESULTS: Initial cell cultures without genetic manipulation underwent senescence within ≤ 15 passages, demonstrating inability to successfully derive primary prostate cancer cell lines. By using SB transposase-based integration of immortalization factors, we were able to establish primary prostate cell lines. Three out of four cell lines displayed epithelial characteristics, however without expression of prostate (cancer) characteristics, e.g., androgen receptor. In vivo, one cell line exhibited tumorigenic potential, yet characteristics of prostate adenocarcinoma were absent. CONCLUSION: Whereas no primary prostate cancer cell line could be established, we provide for the first-time immortalization of primary prostate cells using the SB transposase system, thereby preventing regulatory and molecular issues based on viral immortalization approaches. Although, none of the newly derived cell lines demonstrated prostate cancer characteristics, tumor formation was observed in one cell line. Given the non-prostate adenocarcinoma properties of the tumor, cells have presumably undergone oncogenic transformation rather than prostate cancer differentiation. Still, these cell lines might be used as a tool for research on prostate cancer initiation and early cancer progression.
Assuntos
Células Epiteliais , Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/patologia , Linhagem Celular Tumoral , Animais , Próstata/patologia , Carcinogênese , Telomerase/genética , Transformação Celular NeoplásicaRESUMO
Considering nucleic acids as the language of life and the genome as the instruction manual of cells, their targeted modulation promises great opportunities in treating and healing diseases. In addition to viral gene transfer, the overwhelming power of non-viral mRNA-based vaccines is driving the development of novel gene transporters. Thereby, various nucleic acids such as DNA (pDNA) or RNA (mRNA, siRNA, miRNA, gRNA, or ASOs) need to be delivered, requiring a transporter due to their high molar mass and negative charge in contrast to classical agents. This chapter presents the specific biological hurdles for using nucleic acids and shows how new materials can overcome these.
Assuntos
Nanopartículas , RNA Guia de Sistemas CRISPR-Cas , Humanos , Técnicas de Transferência de Genes , Terapia Genética , RNA MensageiroRESUMO
Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Edição de Genes/métodos , Terapia Genética/métodos , Animais , Vetores Genéticos/genética , Vírus/genéticaRESUMO
X-linked juvenile retinoschisis (XLRS) is an early-onset progressive inherited retinopathy affecting males. It is characterized by abnormalities in the macula, with formation of cystoid retinal cavities, frequently accompanied by splitting of the retinal layers, impaired synaptic transmission of visual signals, and associated loss of visual acuity. XLRS is caused by loss-of-function mutations in the retinoschisin gene located on the X chromosome (RS1, MIM 30083). While proof-of-concept studies for gene augmentation therapy have been promising in in vitro and rodent models, clinical trials in XLRS patients have not been successful thus far. We performed a systematic literature investigation using search strings related to XLRS and gene therapy in in vivo and in vitro models. Three rounds of screening (title/abstract, full text and qualitative) were performed by two independent reviewers until consensus was reached. Characteristics related to study design and intervention were extracted from all studies. Results were divided into studies using (1) viral and (2) non-viral therapies. All in vivo rodent studies that used viral vectors were assessed for quality and risk of bias using the SYRCLE's risk-of-bias tool. Studies using alternative and non-viral delivery techniques, either in vivo or in vitro, were extracted and reviewed qualitatively, given the diverse and dispersed nature of the information. For in-depth analysis of in vivo studies using viral vectors, outcome data for optical coherence tomography (OCT), immunohistopathology and electroretinography (ERG) were extracted. Meta-analyses were performed on the effect of recombinant adeno-associated viral vector (AAV)-mediated gene augmentation therapies on a- and b-wave amplitude as well as the ratio between b- and a-wave amplitudes (b/a-ratio) extracted from ERG data. Subgroup analyses and meta-regression were performed for model, dose, age at injection, follow-up time point and delivery method. Between-study heterogeneity was assessed with a Chi-square test of homogeneity (I2). We identified 25 studies that target RS1 and met our search string. A total of 19 of these studies reported rodent viral methods in vivo. Six of the 25 studies used non-viral or alternative delivery methods, either in vitro or in vivo. Of these, five studies described non-viral methods and one study described an alternative delivery method. The 19 aforementioned in vivo studies were assessed for risk of bias and quality assessments and showed inconsistency in reporting. This resulted in an unclear risk of bias in most included studies. All 19 studies used AAVs to deliver intact human or murine RS1 in rodent models for XLRS. Meta-analyses of a-wave amplitude, b-wave amplitude, and b/a-ratio showed that, overall, AAV-mediated gene augmentation therapy significantly ameliorated the disease phenotype on these parameters. Subgroup analyses and meta-regression showed significant correlations between b-wave amplitude effect size and dose, although between-study heterogeneity was high. This systematic review reiterates the high potential for gene therapy in XLRS, while highlighting the importance of careful preclinical study design and reporting. The establishment of a systematic approach in these studies is essential to effectively translate this knowledge into novel and improved treatment alternatives.
Assuntos
Retinosquise , Masculino , Humanos , Animais , Camundongos , Retinosquise/genética , Retinosquise/terapia , Retinosquise/diagnóstico , Retina/patologia , Eletrorretinografia , Terapia Genética , Mutação , Proteínas do Olho/genéticaRESUMO
BACKGROUND: Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA). METHODS: We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach. RESULTS: Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 108-3.6 × 109 CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity. CONCLUSIONS: Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.
Assuntos
DNA , Linfócitos T , Humanos , Reparo de DNA por Recombinação , Imunoterapia AdotivaRESUMO
The rapid progress of nanotechnology has led to use different nanomaterials for biomedical applications. Among them, graphene-encapsulated magnetic nanoparticles (GEMNS) are recognized as next generation carbon nanomaterials in translation cancer research. In this study, we utilized green fluorescence protein (GFP) expression plasmid DNA (pDNA) and GEMNS decorated with branched polyethyleneimine (PEI) to yield a novel transporter (GEMNS-PEI/pDNA) for gene delivery into melanoma cells (B16F10). The efficiency of transfection was examined using PCR and confocal microscopy. The studies show that the as-designed GEMNS-PEI construct is successfully used to transfect the melanoma cells with pDNA and it should be considered as a potent non-viral vector for introducing naked nucleic acids into eucaryotic cells.